α-Costic acid is a natural sesquiterpene possessing diverse biological activities. Among these, the most promising property for practical application is its acaricidal activity against Varroa destructor, considered the most important and dangerous parasite of the honeybee (Apis mellifera L.). Infestations of V. destructor can decimate bee populations in just a few years, resulting in substantial environmental and economic losses. This study reports the synthesis of α- and γ-costic acid diesters with ethylene glycol and evaluates their acaricidal efficacy against V. destructor in comparison with their parent acids. Although the resulting dimers were approximately 50% less active than the parent costic acids, they exhibited higher potency than the previously reported α-costic acid methyl ester. Among all, γ-costic acid, whose acaricidal activity is reported here for the first time, shows the highest activity with mortality higher than 90%, thus emerging as the most promising candidate for the development of effective and environmentally friendly strategies for the control of V. destructor. Furthermore, it may hold potential for the biocontrol of other mite pests affecting economically important crops such as legumes and cereals, which are currently associated with significant yield losses and extensive reliance on synthetic pesticides.

In this study, we characterised and compared the metabolites of multifloral samples of bee pollen (BP) and bee bread (BB) collected from two A. mellifera ligustica apiaries located at different altitudes in the Campania region (southern Italy): the Castelpoto Valley (BPv and BBv) and the Taburno Massif (BPm and BBm). Untargeted LC-MS/MS profiling enabled the identification of 50 phenolamides (mainly tri-caffeoyl, tri-coumaroyl, and caffeoyl-di-coumaroyl conjugates of spermidine and spermine) and 16 glycosylated flavonoids (primarily derivatives of isorhamnetin, quercetin, and kaempferol). Exploratory data analysis using Partial Least Squares showed that metabolites significantly vary according to both geographical site and matrix (BP vs BB). BPv had higher levels of di-p-coumaroyl spermidine and tetra-p-coumaroyl spermine isomers, while BPm and BBv were richer in tri-p-coumaroyl spermidine isomers.Pollen botanical origin was identified by Internal Transcribed Spacer 2 (ITS2) metabarcoding with Illumina Next-Generation Sequencing (NGS), which revealed significant differences in botanical composition between the collection sites and between BP and BB. BPv predominantly derived from Asteraceae and Fabaceae, while BBv samples were characterized by prevalent Fagaceae (mainly Castanea sativa) origin. BPm samples originated primarily from Boraginaceae and Fabaceae, while BBm samples were strongly dominated by Castanea sativa and Rosaceae.Overall, this study defines how geographic and botanical origin influence the content of bioactive compounds in BP and BB, highlighting the potential use of phenolic profiles as chemotaxonomic fingerprints and indicators of nutritional quality.

In recent years, persistent environmental contaminants such as PFAS have emerged alongside traditional pollutants like heavy metals. Honey is a useful biomonitoring tool thanks to bees' foraging range, which reflects average pollution levels. The objective of this study was to determinate heavy metals and PFAS in honey from six areas of the Molise region (rural, industrial, and remote), to evaluate environmental impact on the matrix, verify if honey meets health standards, and assess potential consumer risks. 56 honey samples were collected over four months directly from honeycombs. Heavy metals were analyzed by ICP-AES; PFAS by SPE-LC-MS/MS. Aluminium, antimony, and selenium were the most detected metals; three PFAS (Bistriflimide, 6:2FTS, L-PFOS) were found at ultra-trace levels. Chemometric analysis identified arsenic, antimony, and selenium as area-specific markers. Risk assessment showed higher non-carcinogenic risk in areas A and E, and carcinogenic risk in C and D. Area B was the least impacted.

The honey bee gut microbiota plays a crucial role in host nutrition, immunity, and colony health, yet the relative influence of seasonal and colony-specific factors on its long-term dynamics remains incompletely understood. This study investigated temporal variation in the gut bacterial community of three Apis mellifera ligustica colonies maintained in the same apiary and monitored over two consecutive years (2022–2023). Worker bees were sampled during eight seasonal periods, and gut microbiota composition was characterized using 16S rRNA gene amplicon sequencing. Across all sampling periods, the microbiome was consistently dominated by the characteristic honey bee-associated genera Gilliamella, Snodgrassella, Bartonella, Frischella, Commensalibacter, and Lactobacillus, indicating the persistence of a conserved core bacterial community. Seasonal variation was primarily associated with changes in the relative abundance of dominant taxa rather than with major changes in community composition. In particular, Gilliamella apicola and Snodgrassella alvi exhibited complementary seasonal patterns, with Gilliamella reaching its highest abundance during autumn, particularly in autumn 2023, whereas Snodgrassella predominated during spring and winter. Alpha-diversity metrics (Observed OTUs, Chao1, Shannon, and Simpson indices) showed limited seasonal variation, whereas beta-diversity analyses detected significant differences in community composition among seasons. Principal Coordinates Analysis and PERMANOVA identified season as the factor most strongly associated with microbiome variation, while colony identity did not significantly influence bacterial community composition under the standardized experimental conditions adopted in this study. Overall, these findings show that the gut microbiome of A. mellifera ligustica maintains a conserved core bacterial community while exhibiting reproducible seasonal variation in the relative abundance of its dominant members. This study provides a longitudinal baseline for future investigations aimed at understanding the ecological mechanisms underlying seasonal microbiome dynamics and their relationship with honey bee biology and environmental change.

Chalkbrood is the manifestation of the fungal disease caused by Ascosphaera apis, which affects broods of developing honeybees, particularly in Apis mellifera colonies. Recently, Metschnikowia pulcherrima has been proposed as a biocontrol agent in winemaking and for the management of major postharvest and soil-borne plant pathogenic fungi. In this study, the antagonistic activity of three M. pulcherrima strains against fifteen A. apis strains, isolated from contaminated hives of A. mellifera, was evaluated, with a specific focus on the potential antifungal activity of volatile organic compounds (VOCs). The study revealed that M. pulcherrima was effective against A. apis and that the antifungal activity was related to various mechanisms including competition for nutrients, secretion of pulcherriminic acid, and biosynthesis of specific antifungal VOCs. We also found that each M. pulcherrima strain produced a unique combination of VOCs, and the antifungal activity was strain-dependent and varied depending on the specific yeast-mold combination. In addition, preliminary analyses showed that a temperature of 30 °C and a higher amount of glucose (40 g/L) in the growing medium promote the growth of A. apis. These results could be useful for designing new strategies for the biocontrol of chalkbrood disease in honeybee colonies.

IntroductionThe genus Bifidobacterium is a key component of the honey bee gut microbiota, playing a fundamental role in maintaining host health and colony well-being. Alongside other core genera such as Bombilactobacillus, Gilliamella, Lactobacillus, and Snodgrassella, Bifidobacterium contributes to essential functions including nutrient digestion, immune modulation, and protection against pathogens. Among threats to honey bee health, Chalkbrood disease, caused by fungus Ascosphaera apis, remains a major concern due to detrimental effects on colony strength and honey yield.Materials and methodsWe characterized enzymatic activity and carbohydrate assimilation of nine Bifidobacterium strains isolated from the honey bee intestinal tract. In parallel, we assessed antifungal potential against A. apis strains, focusing on volatile organic compounds (VOCs).Results and discussionNotably, Bifidobacterium asteroides 3CP-2B exhibited enzymatic capabilities supporting digestive functions and metabolism of sugars potentially harmful to honey bees. This strain showed marked antifungal activity against A. apis, mediated by volatile and non-volatile bioactive metabolites. Among VOCs identified, propanoic acid, ethanol, acetic acid, ethyl propionate, and 1-propanol were the most prominent compounds associated with the antifungal effect.

Introduction:Among probiotics, Lactic Acid Bacteria modulate host immunity via strain-specific molecular patterns. The invertebrate model Galleria mellonella offers conserved innate immune pathways and is increasingly applied for preclinical screening of probiotic functions. Methods:We evaluated the immunomodulatory activity of Lactiplantibacillus plantarum ATCC 14917 and Lentilactobacillus kefiri DSM 10551 in G. mellonella. Larvae were injected with 106 CFU/larva of each strain, and survival and health indices were monitored for 72 h. The temporal transcriptional response of ten innate immunity-related genes, including Toll and IMD signalling, Toll receptor, cytokine-like ligand, and antimicrobial effectors, was assessed via qRT-PCR over 3-24 hours, complemented by correlation and hierarchical clustering to identify co-expression modules and strain-specific transcriptional patterns. Results:Both strains were non-toxic and induced strain-dependent gene expression patterns. L. plantarum induced a stronger and more sustained activation of immune signalling pathways and effector responses, whereas L. kefiri was characterised by an earlier and prolonged activation of stress-related and tissue-protective mechanisms. Correlation and clustering analyses revealed distinct co-expression modules that reflect modulation of the Toll and IMD pathways. Discussion:These findings suggest that G. mellonella could serve as a cost-effective in vivo model for functional screening of Lactobacillus spp. with immunomodulatory potential and possible translational relevance to human innate immunity.

Xylella fastidiosa, a notorious bacterial plant pathogen with a broad host range, has recently emerged as a significant threat to olive trees worldwide, causing the Olive Quick Decline Syndrome (OQDS). This disease severely affects olive production and leads to the death of the plants, causing significant economic losses and dramatic changes in the landscape. To date, no determined control strategies have been identified to fight the OQDS. Since the pathogen is an obligate host of some species of spittlebugs, managing the insect vectors themselves may be an effective approach for limiting OQDS. For that, plant-based products, due to their low toxicity to non-target organisms and minimal adverse effects on the environment, have gained significant interest as tools to tackle the diffusion of OQDS through X. fastidiosa insect vector management. Ongoing studies are exploring the role of plant secondary metabolites as infochemicals in insect–host plant interactions. Some of these focus on essential oils, plant extracts, and their major constituents, examining their potential role as toxic or attractive/repellent active ingredients against insects. These compounds are being evaluated as bioinsecticides or in semiochemical-based control strategies (e.g. mass trapping, push–pull, attract-and-kill) potentially relevant for monitoring and controlling the meadow spittlebug Philaenus spumarius (L.), the primary vector of X. fastidiosa subspecies pauca. In this scenario, our review offers an analysis of the current state of research on botanical products applied as toxicants or behaviour-modifying tools towards P. spumarius, highlighting emerging developments, innovative technologies, and recent breakthroughs.

BACKGROUND Xylella fastidiosa is an important causative agent of Olive Quick Decline Syndrome in the Apulia region of Italy. The current study evaluated the bioefficacy of three entomopathogenic fungal strains: Beauveria bassiana SGB7004, Metarhizium robertsii SGB1K, and Akanthomyces lecanii SGB4711 against Philaenus spumarius the main vector of this pathogen, under laboratory conditions. Pathogenicity bioassays were performed by dipping nymphs and adults of P. spumarius in an aqueous suspension of powdered fungal culture (PFC) or conidial suspension (CS) of the three fungal strains. RESULTS Both B. bassiana SGB7004 and M. robertsii SGB1K affected the viability of nymphs, resulting in more than 80% mortality at 48 h post treatment, while the effect of A. lecanii SGB4711 was not statistically significant. On adults, all three biocontrol strains were effective in a time- and concentration-dependent manner. The PFCs of B. bassiana SGB7004, M. robertsii SGB1K, and A. lecanii SGB4711 at the highest concentration tested (120 mg mL(-1)) resulted in 97%, 83% and 27% mortality at the trial endpoint (120 h), respectively. Mycelial growth was observed on 38.5%, 37.0% and 61.5% of dead insects treated with B. bassiana SGB7004 (2.3 x 10(8) CFU mL(-1)), M. robertsii SGB1K (3.8 x 10(6) CFU mL(-1)) and A. lecanii SGB4711 (5.4 x 108 CFU mL(-1)), respectively. None of the PFCs of the tested strains was pathogenic when injected into nymph spittle. CONCLUSIONS Beauveria bassiana SGB7004 and M. robertsii SGB1K significantly affected the survival of P. spumarius nymphs and adults, while A. lecanii SGB4711 was not effective on nymphs and only slightly effective against adults.

The pollination of several crops, as well as wild plants, depends on honeybees. To get the nutrients required for growth and survival, honeybee colonies are dependent on pollen supply. Bee pollen (BP) is partially packed in honeycomb cells and processed into beebread (BB) by microbial metabolism. The composition of pollen is highly variable and is mainly dependent on ecological habitat, geographical origin, honey plants, climatic conditions, and seasonal variations. Although there are important differences between the BP and the BB, little comparative chemical and microbiological data on this topic exists in the literature, particularly for samples with the same origin. In this study, BP and BB pollen samples were collected from two apiaries located in the Campania and Molise regions of Southern Italy. Phenolic profiles were detected via HPLC, while antioxidant activity was determined by ABTS·+ and DPPH· assay. The next-generation sequencing (NGS) based on RNA analysis of 16S (rRNA) and internal transcribed spacer (ITS2) regions were used to investigate the microbial community (bacteria and fungi) and botanical origin of the BP and BB. Chemical analysis showed a higher content of flavonols in BP (rutin, myricetin, quercetin, and kaempferol), while in BB there was a higher content of phenolic acids. The NGS analysis revealed that the microbial communities and pollen sources are dependent on the geographical location of apiaries. In addition, diversity was highlighted between the microbial communities present in the BP and BB samples collected from each apiary.

BACKGROUND The meadow spittlebug Philaenus spumarius L. is the vector for the bacterium Xylella fastidiosa subspecies pauca, involved in "Olive Quick Decline Syndrome" in Salento (Italy). Control of P. spumarius is key to limiting transmission of the bacterium, and an innovative approach can be based on effective natural compounds and biocontrol agents. Entomopathogenic fungi are an important source of bioactive natural molecules that play a role in the relationship between microorganisms and insects. RESULTS Pathogenicity bioassays, performed by dipping adults of P. spumarius in either fungal culture suspension (120 mg/mL) or cell-free culture supernatant of Trichoderma chlorosporum GJS 91-150 showed respectively 97 % and 87 % death, within 24 hours. The effect was dose-dependent. In laboratory bioassays, the powdered fungal culture of T. chlorosporum GJS 91-150 did not exhibit pathogenic activity when injected into nymph spittle. CONCLUSIONS T. chlorosporum GJS 91-150 affected the survival of P. spumarius adults. The lethal effect was not associated with the development of mycelium on the cuticle, but seems due, at least partly, to fungal metabolites released in the culture medium. The fungus tested here has a good potential for the development of effective low-environmental impact control strategies for P. spumarius and suppression of X. fastidiosa. This article is protected by copyright. All rights reserved.

Polycyclic aromatic hydrocarbons (PAHs), heavy metals, and plasticizer residues are continuously released into the environment. The use of living organisms, such as Apis mellifera L. and honey, is advantageous as bioindicator of the environmental health status, instead of traditional monitoring methods, showing the ability to record spatial and temporal pollutant variations. The PAHs and heavy metal presence were determined in two sampling years (2017 and 2018) in five different locations in the Molise region (Italy), characterized by different pollution levels. During 2017, most PAHs in all samples were lower than limit of detection (LOD), while in 2018, their mean concentration in bee and honey samples was of 3 μg kg−1 and 35 μg kg−1, respectively. For heavy metals, lower values were detected in 2017 (Be, Cd, and V below LOD), while in 2018, the mean concentrations were higher, 138 μg kg−1 and 69 μg kg−1, in bees and honey, respectively. Honey has been used as indicator of the presence of phthalate esters and bisphenol A in the environment. The satisfactory results confirmed that both bees and honey are an important tool for environmental monitoring. The chemometric analysis highlighted the differences in terms of pollutant concentration and variability in the different areas, validating the suitability of these matrices as bioindicators.

Nosemosis is a disease triggered by the single-celled spore-forming fungi Nosema apis and Nosema ceranae, which can cause extensive colony losses in honey bees (Apis mellifera L.). Fumagillin is an effective antibiotic treatment to control nosemosis, but due to its toxicity, it is currently banned in many countries. Accordingly, in the beekeeping sector, there is a strong demand for alternative ecological methods that can be used for the prevention and therapeutic control of nosemosis in honey bee colonies. Numerous studies have shown that plant extracts, RNA interference (RNAi) and beneficial microbes could provide viable non-antibiotic alternatives. In this article, recent scientific advances in the biocontrol of nosemosis are summarized.

The increased mobility of goods, people, and animals worldwide has caused the spread of several arthropod vectors, leading to an increased risk of animal and human infections. Aedes koreicus is a common species in South Korea, China, Japan, and Russia. Due to its cold-resistant dormant eggs, the adults last from the late summer until the autumn seasons. For these reasons, it seems to be better adapted to colder temperatures, favoring its colonization of hilly and pre-alpine areas. Its first appearance in Europe was in 2008 in Belgium, where it is currently established. The species was subsequently detected in Italy in 2011, European Russia, Germany, the Swiss–Italian border region, Hungary, Slovenia, Crimea, Austria, the Republic of Kazakhstan, and the Netherlands. The role of A. koreicus in the transmission of vector-borne pathogens remains unclear. The available scientific evidence is very old, often not available in English or not indexed in international databases, and therefore difficult to find. According to the literature reviewed, A. koreicus can be considered a new invasive mosquito species in Europe, establishing populations on the European continent. In addition, experimental evidence demonstrated its vector competence for both Dirofilaria immitis and Chikungunya and is relatively low for ZIKA but not for Western Nile Virus. On the other hand, even if the field evidence does not confirm the experimental findings, it is currently not possible to exclude with absolute certainty the potential involvement of this species in the spread, emergence, or re-emergence of these vector-borne disease agents.

The meadow spittlebug, Philaenus spumarius L. (Hemiptera: Auchenorrhyncha: Aphrophoridae), is the main vector of Xylella fastidiosa subsp. pauca strain ST53, the causal agent of the Olive Quick Decline Syndrome. Philaenus spumarius and other Auchenorrhyncha are known to communicate via vibrations, whereas the possible occurrence of semiochemical communication has been poorly investigated so far. Through a chemical ecology approach, we provide evidence of intraspecific chemical communication in P. spumarius. In Y-tube olfactometer bioassays, males were attracted to unmated females as well as toward the headspace volatile extracts collected from unmated females. Conversely, females did not respond to unmated male volatiles or their extracts, nor did males and females respond to volatiles from individuals of the same sex. Electroantennography assays of unmated male and female headspace extracts elicited measurable responses in the antennae of both sexes. Male responses to body wash extracts from both sexes were stronger compared to female responses. Thus, suggesting the presence of compounds that are highly detected by the male's olfactory system. The female head seemed to be the source of such compounds. This is the first record of intraspecific chemical communication in P. spumarius and one of the very few records in Auchenorrhyncha. Possible biological roles are under investigation.

Honey bees (Apis mellifera) are agriculturally important pollinators. Over the past decades, significant losses of wild and domestic bees have been reported in many parts of the world. Several biotic and abiotic factors, such as change in land use over time, intensive land management, use of pesticides, climate change, beekeeper's management practices, lack of forage (nectar and pollen), and infection by parasites and pathogens, negatively affect the honey bee's well-being and survival. The gut microbiota is important for honey bee growth and development, immune function, protection against pathogen invasion; moreover, a well-balanced microbiota is fundamental to support honey bee health and vigor. In fact, the structure of the bee's intestinal bacterial community can become an indicator of the honey bee's health status. Lactic acid bacteria are normal inhabitants of the gastrointestinal tract of many insects, and their presence in the honey bee intestinal tract has been consistently reported in the literature. In the first section of this review, recent scientific advances in the use of LABs as probiotic supplements in the diet of honey bees are summarized and discussed. The second section discusses some of the mechanisms by which LABs carry out their antimicrobial activity against pathogens. Afterward, individual paragraphs are dedicated to Chalkbrood, American foulbrood, European foulbrood, Nosemosis, and Varroosis as well as to the potentiality of LABs for their biological control.

The balance of the gut microbiome is important for the honey bee's growth and development, immune function and defense against pathogens. The use of a beneficial bacteria-based strategy for the prevention and biocontrol of American foulbrood (AFB) and European foulbrood (EFB) diseases in honey bees offers interesting prospects. Lactic acid bacteria (LAB) are common inhabitants of the gastrointestinal tract of the honey bee. Among LABs associated with bee gut microbiota, Lactiplantibacillus plantarum (previously Lactobacillus plantarum) and Apilactobacillus kunkeei (formerly classified as Lactobacillus kunkeei) are two of the most abundant species. In this study, four Lactiplantibacillus plantarum strains and four Apilactobacillus kunkeei strains, isolated from the gastrointestinal tract of honey bee (Apis mellifera L.) were selected for their in vitro inhibition ability of Paenibacillus larvae ATCC 9545 and Melissococccus plutonius ATCC 35311. In addition, these LABs have been characterized through some biochemical and functional characteristics: cell surface properties (hydrophobicity and auto-aggregation), carbohydrates assimilation and enzymatic activities. The antimicrobial, biochemical and cell surface properties of these LABs have been functional to their candidature as potential probiotics in beekeeping and for the biocontrol of AFB and EFB diseases.

Ascosphaera apis is an entomopathogenic fungus that affects honeybees. In stressful conditions, this fungus (due not only to its presence, but also to the combination of other biotic and abiotic stressors) can cause chalkbrood disease. In recent years, there has been increasing attention paid towards the use of lactic acid bacteria (LAB) in the honeybees' diets to improve their health, productivity and ability to resist infections by pathogenic microorganisms. The screening of 22 strains of Lactiplantibacillus plantarum, isolated from the gastrointestinal tracts of honeybees and beebread, led to the selection of five strains possessing high antagonistic activity against A. apis. This study focused on the antifungal activity of these five strains against A. apis DSM 3116 and DSM 3117 using different matrices: cell lysate, broth culture, cell-free supernatant and cell pellet. In addition, some functional properties and the antioxidant activity of the five L. plantarum strains were evaluated. All five strains exhibited high antagonistic activity against A. apis, good surface cellular properties (extracellular polysaccharide (EPS) production and biofilm formation) and antioxidant activity. Although preliminary, these results are encouraging, and in future investigations, the effectiveness of these bacteria as probiotics in honeybee nutrition will be tested in vivo in the context of an eco-friendly strategy for the biological control of chalkbrood disease.

(1) Monitoring of Drosophila suzukii is based on the use of effective traps and baits. The current baits are insufficient to provide efficient monitoring. The use of bacteria as bio-catalyzers to produce bioactive volatiles may improve flies' attraction. Thus, we conducted this work to improve Droskidrink® bait's attractiveness using lactic acid bacteria. (2) Different baits that were based on the use of Droskidrink® were assessed for flies' attraction in a Droso-Trap® in a vineyard. Oenococcus oeni, Pediococcus spp., and Lactobacillus spp. were used. The performance of the most attractive species, O. oeni, inoculated into Droskidrink® was assessed in laboratory tests. The responses of female flies to volatiles produced by Droskidrink® with O. oeni strains were recorded by electroantennography. (3) Preliminary field assessment of baits recorded O. oeni as the most attractive species. Three strain groups showed adaptation to test conditions. Volatiles extracted by the headspace of baits inoculated with O. oeni, elicited electroantennographic responses from fly antennae. (4) Droskidrink® inoculated with O. oeni is a highly attractive bait for monitoring. These findings will be useful for improving the attractiveness of D. suzukii commercial baits based on the utilization of LAB volatiles in a strain-dependent manner.

Aedes albopictus (Skuse) is a widespread mosquito, a vector of important human arboviruses, including Chikungunya, Dengue and Zika. It is an extremely difficult species to control even for the onset of resistances to chemicals insecticides, therefore ecofriendly products are urgently needed. In this study, the activity of Amaryllidaceae alkaloids and some of their semisynthetic derivatives, of 2-methoxy-1,4-naphthoquinone and two analogues, of cyclopaldic acid and epi-epoformin on the survival and development of Ae. albopictus larvae was evaluated. First-instar larval exposure for 24 and 48 h to cyclopaldic acid, resulted in mortality mean per-centage of 82.4 and 96.9 respectively; 1,2-O,O-diacetyllycorine 48h post-treatment caused 84.7% mortality. Larval and pupal duration were proved to decrease significantly when larvae were exposed to cyclopaldic acid, 1,2-O,O-diacetyllycorine and N-methyllycorine iodide. The mean number of third-instar larvae surviving to 2-methyl-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone and 2-methoxy-1,4-naphthoquinone was significantly lower than the number of correspondent control larvae over the time. This study indicated that 1,2-O,O’-diacetyllycorine, N-methyllycorine iodide, cyclopaldic acid and 1,4-naphthoquinone structural derivatives have good potential for developing bioinsecticides for mosquito control programs. The obtained results are of general interest due to the global importance of the seri-ous human diseases such a vector is able to spread.