Multi-Elemental Analysis as a Tool to Ascertain the Safety and the Origin of Beehive Products: Development, Validation, and Application of an ICP-MS Method on Four Unifloral Honeys Produced in Sardinia, Italy.

Despite unifloral honeys from Sardinia, Italy, being appreciated worldwide for their peculiar organoleptic features, their elemental signature has only partly been investigated. Hence, the principal aim of this study was to measure the concentration of trace and toxic elements (i.e., Ag, As, Ba, Be, Bi, Cd, Co, Cr, Cu, Fe, Hg, Li, Mn, Mo, Ni, Pb, Sb, Sn, Sr, Te, Tl, V, and Zn) in four unifloral honeys produced in Sardinia. For this purpose, an original ICP-MS method was developed, fully validated, and applied on unifloral honeys from asphodel, eucalyptus, strawberry tree, and thistle. Particular attention was paid to the method's development: factorial design was applied for the optimization of the acid microwave digestion, whereas the instrumental parameters were tuned to minimize the polyatomic interferences. Most of the analytes' concentration ranged between the relevant LoDs and few mg kg-1, while toxic elements were present in negligible amounts. The elemental signatures of asphodel and thistle honeys were measured for the first time, whereas those of eucalyptus and strawberry tree honeys suggested a geographical differentiation if compared with the literature. Chemometric analysis allowed for the botanical discrimination of honeys through their elemental signature, whereas linear discriminant analysis provided an accuracy level of 87.1%.

Honeybees use propolis as a natural pesticide against their major ectoparasite.

Honeybees use propolis collected from plants for coating the inner walls of their nest. This substance is also used as a natural antibiotic against microbial pathogens, similarly to many other animals exploiting natural products for self-medication. We carried out chemical analyses and laboratory bioassays to test if honeybees use propolis for social medication against their major ectoparasite: Varroa destructor. We found that propolis is applied to brood cells where it can affect the reproducing parasites, with a positive effect on honeybees and a potential impact on Varroa population. We conclude that propolis can be regarded as a natural pesticide used by the honeybee to limit a dangerous parasite. These findings significantly enlarge our understanding of behavioural immunity in animals and may have important implications for the management of the most important threat to honeybees worldwide.

Fiber Bragg Grating Sensors for Underwater Vibration Measurement: Potential Hydropower Applications.

The present paper assesses the performance and characteristics of fiber Bragg grating sensors, with a special interest in their applications in hydraulic machinery and systems. The hydropower industry is turning to this technology with high expectations of obtaining high quality data to validate and calibrate numerical models that could be used as digital twins of key assets, further strengthening the sector's relevant position within industry 4.0. Prior to any validation, fiber Bragg grating sensors' ability to perform well underwater for long periods of time with minimal degradation, and their ease of scalability, drew the authors´ attention. A simplified modal analysis of a partially submerged beam is proposed here as a first step to validate the potential of this type of technology for hydropower applications. Fiber Bragg grating sensors are used to obtain the beam's natural frequencies and to damp vibrations under different conditions. The results are compared with more established waterproof electric strain gauges and a laser vibrometer with good agreement. The presence of several sensors in a single fiber ensures high spatial resolution, fundamental to precisely determine vibration patterns, which is a main concern in this industry. In this work, the beam's vibration patterns have been successfully captured under different excitations and conditions.

Electrochemical Determination of the "Furanic Index" in Honey.

5-(hydroxymethyl)furan-2-carbaldehyde, better known as hydroxymethylfurfural (HMF), is a well-known freshness parameter of honey: although mostly absent in fresh samples, its concentration tends to increase naturally with aging. However, high quantities of HMF are also found in fresh but adulterated samples or honey subjected to thermal or photochemical stresses. In addition, HMF deserves further consideration due to its potential toxic effects on human health. The processes at the origin of HMF formation in honey and in other foods, containing saccharides and proteins-mainly non-enzymatic browning reactions-can also produce other furanic compounds. Among others, 2-furaldehyde (2F) and 2-furoic acid (2FA) are the most abundant in honey, but also their isomers (i.e., 3-furaldehyde, 3F, and 3-furoic acid, 3FA) have been found in it, although in small quantities. A preliminary characterization of HMF, 2F, 2FA, 3F, and 3FA by cyclic voltammetry (CV) led to hypothesizing the possibility of a comprehensive quantitative determination of all these compounds using a simple and accurate square wave voltammetry (SWV) method. Therefore, a new parameter able to provide indications on quality of honey, named "Furanic Index" (FI), was proposed in this contribution, which is based on the simultaneous reduction of all analytes on an Hg electrode to ca. -1.50 V vs. Saturated Calomel Electrode (SCE). The proposed method, validated, and tested on 10 samples of honeys of different botanical origin and age, is fast and accurate, and, in the case of strawberry tree honey (Arbutus unedo), it highlighted the contribution to the FI of the homogentisic acid (HA), i.e., the chemical marker of the floral origin of this honey, which was quantitatively reduced in the working conditions. Excellent agreement between the SWV and Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) data was observed in all samples considered.

Safety evaluation of the entomopathogenic bacterium Brevibacillus laterosporus for the green lacewing Chrysoperla agilis (Neuroptera: Chrysopidae).

The safety of the entomopathogenic bacterium Brevibacillus laterosporus for the natural insect predator Chrysoperla agilis was evaluated in this study. For this purpose, laboratory bioassays were conducted exposing different larval instars and adults of the chrysopid to bacterial spore preparations, in order to evaluate the possible effects on survival, longevity, immature development, and adult reproductive performance. The sub-lethal effects were investigated by feeding the bacterium directly to adults and larvae of C. agilis or to mealworm beetles (Tenebrio molitor) used as hosts for chrysopids (tritrophic interaction). Direct feeding of B. laterosporus spores to different lacewing larvae instars and to adults did not cause mean mortality levels significantly different from untreated control, and slight though not significant effects of treatments were generally observed on insect longevity, development, fecundity and egg hatching. In the case of lacewing larvae feeding on treated mealworm beetles, adult emergence percentage was reduced approximately 12%, in comparison with untreated control. Based on these results, the use of B. laterosporus for pest management in the agroecosystem, appears to be compatible with chrysopids.

Temperature-insensitive optical tilt sensor based on a single eccentric-core fiber Bragg grating.

This Letter presents a simple temperature-insensitive optical tilt sensor based on a single eccentric-core fiber Bragg grating (ECFBG). By partly inserting an ECFBG into a ceramic ferrule, the reflection spectrum of the ECFBG splits into two peaks as a result of the applied tilt angle. The magnitude and direction of inclination in one dimension can be determined by monitoring the wavelength separation between both peaks, which is inherently insensitive to temperature. The proposed tilt sensor has a good linear response within a wide dynamic range of ±45°, with a sensitivity of 0.012 nm/°, with a resolution of 0.83°, and with an accuracy of 0.41°. Being free from any inherent mechanical joint/friction, along with the advantages of a compact structure, good repeatability, and low cost, the proposed sensor is highly suitable for practical engineering applications.

The effects of raw propolis on Varroa-infested honey bee (Apis mellifera) workers.

Self-medication plays a major role in the behavioral defense against pathogens and parasites that animals have developed during evolution. The conditions defining this adaptive behavior are: (1) contact with the substance in question must be deliberate; (2) the substance must be detrimental to one or more parasites; (3) the detrimental effect on parasites must lead to increased host fitness. Recent studies have shown that A. mellifera colonies are able to increase resin foraging rates when infested by V. destructor, whereas further investigations are needed for evidence of parasite and host fitness. In order to understand whether Varroa-infested colonies could benefit from increasing levels of resin, we carried out laboratory bioassays to investigate the effects of propolis on the fitness of infested workers. The longevity and energetic stress of adult bees kept in experimental cages and artificially infested with the mite were thus monitored over time. At the same time, in vitro experiments were performed to study the contact effects of crude propolis on Varroa mites. Our results clearly demonstrate the positive effects of raw propolis on the lifespan of Varroa-infested adult bees. A low narcoleptic effect (19-22%) of raw propolis on phoretic mites after 5 h was also observed. In terms of energetic stress, we found no differences between Varroa-free and Varroa-infested bees in terms of the daily sucrose solution demand. Our findings seem to confirm the hypothesis that resin collection and propolis use in the hive represent an example of self-medication behavior in social insects.

Pathogenicity and characterization of a novel Bacillus cereus sensu lato isolate toxic to the Mediterranean fruit fly Ceratitis capitata Wied.

The lethal and sub-lethal effects of sporulated cultures of a novel Bacillus cereus sensu lato strain lacking detectable cry genes and identified through morphological and genetic analyses, have been studied on the Mediterranean fruit fly Ceratitis capitata. The lethal effects on young larvae were concentration dependent, with a median lethal concentration (LC50) of 4.48 × 10(8)spores/g of diet. Sporulated cultures of this strain significantly extended development time and reduced immature survival, and the size of emerging fly adults. Besides spores, the toxicity has been associated to the insoluble extra-spore fraction characterized through a proteomic approach. The profile of the extra-spore protein fraction (ES) showed major protein bands within the 35-65 kDa range. The results of mass spectrometry analysis highlighted the presence of putative virulence factors, including members of protein families previously associated to the insecticidal action of other microbial entomopathogens. These proteins include metalloproteases, peptidases and other enzymes.

Observations on house fly larvae midgut ultrastructure after Brevibacillus laterosporus ingestion.

The pathological and histopathological course caused by Brevibacillus laterosporus on house fly larvae has been investigated conducting observations on insect behavior and midgut ultrastructure. After dissection and fixation, gut tissues were analyzed under transmission electron microscopy (TEM) in order to compare in vivo-treated and non-treated (control) fly specimens. Treated larvae showed extensively reduced feeding and growth rate, then became sluggish and died within 72 h. A progressive midgut epithelium deterioration was observed in treated larvae, compared to the control. Ultrastructural changes consisted of microvilli disruption, cytoplasm vacuolization and general disorganization, endoplasmic reticulum deformation, mitochondria alteration. Deterioration became progressively more dramatic until the infected cells released their content into the gut lumen. Disruption was associated also with midgut muscular sheath and connective tissue. These ultrastructural changes are similar to those widely described for other entomopathogenic bacteria, such as Bacillus thuringiensis, against different insect species. The rapid disruption of cellular fine structure supports a hypothesis based on an interaction of toxins with the epithelial cell membranes reminiscent of the specific B. thuringiensis δ-endotoxins mechanism of action on other insect targets.

Bio-Ethology of Vespa crabro in Sardinia (Italy), an Area of New Introduction.

Vespa crabro, also known as European hornet, is a eusocial Vespidae originally from Eurasia that was accidentally introduced on the island of Sardinia (Italy) in 2010. Currently, its distribution is limited to the northern area of the island. Considering that a non-harmful species in its native region can exhibit invasive behaviour when established in new environments, bio-ethological observations were conducted to better understand whether V. crabro could show invasive traits in Sardinia, which represents a new introduction area. Data on the foraging activity of the European hornet in open fields were collected within a citizen science monitoring program carried out in Sardinia from 2018 to 2020. Moreover, specific behavioural observations were made in apiaries to assess the predatory activity of the hornet towards honey bees and at the entrance of free-living European hornet colonies to evaluate other aspects of its behaviour, i.e., intranidal and extranidal tasks. The results of our study are discussed in relation to the behavioural traits known for this species in its native areas to place the behavioural repertoire of V. crabro in Sardinia into a wider context. Our observations revealed that V. crabro did not show any changes in behavioural traits in Sardinia compared to those described in its area of origin, so the risk of becoming an invasive species on this island seems unlikely.

The Sardinian Bitter Honey: From Ancient Healing Use to Recent Findings.

Sardinian bitter honey, obtained from the autumnal flowering of the strawberry tree (Arbutus unedo L.), has an old fame and tradition in popular use, especially as a medicine. Its knowledge dates back over 2000 years, starting from the Greeks and Romans to the present day. There are many literary references from illustrious personalities of the past such as Cicero, Horace, Virgil, and Dioscorides, until recent times, associated with the peculiar anomaly of its taste, which lends itself to literary and poetic metaphors. The curiosity of its bitter taste is also what led to the first studies starting in the late 1800s, aimed to reveal its origin. Other studies on its botanical source and characteristics have been carried out over time, up to the most recent investigations, which have confirmed its potential for use in the medical field, thanks to its antioxidant, antiradical, and cancer-preventing properties. These benefits have been associated with its phenolic component and in particular with the prevailing phenolic acid (homogentisic acid). Later, other strawberry tree honeys from the Mediterranean area have also shown the same properties. However, Sardinian bitter honey maintains its geographical and historical identity, which is recognized by other Mediterranean cultures.

Where Is the Honey Bee Queen Flying? The Original Case of a Foraging Queen.

During a bee fauna survey in the countryside of northern Sardinia, a honey bee queen (Apis mellifera L.) was detected while foraging on a borage (Borago officinalis L.) flower in Uri, Province of Sassari, Italy, most likely during an orientation flight before mating. Morphological details, detectable from photos with the naked eye and stereomicroscopic observations, confirmed that the honey bee queen was sucking nectar from a flower. The enormous development of the abdomen, lack of pollen-collecting structures in the legs and other characteristics such as the typical distally bilobed shape of the mandibles, with long hairs on their outer surface, proved the structural differences between the queen specimen and the other castes of bees. The queen's proboscis, which is shorter compared to the workers, may have been counterbalanced by the shape and nectar production of the borage flower. This new observation proves that the queen can feed herself under natural conditions, likely to obtain the energy required for flying. Although we cannot exclude disturbing factors that could explain this foraging behaviour of a queen observed for the first time, this note opens a new scenario and discusses this new finding in the context of the available literature on the queen's behaviour and questions to be answered.

In Vitro Activity of Several Essential Oils Extracted from Aromatic Plants against Ascosphaera apis.

The use of natural substances such as essentials oils against bee pathogens is of great interest as an alternative to traditional methods based on synthetic compounds like antibiotics and fungicides, in order to minimize the risk of having toxic residues in hive products and to prevent the development of resistance phenomena. This study evaluated the inhibitory, fungicidal and sporicidal activity of ten essential oils extracted from aromatic plants against Ascosphaera apis, the etiological agent of chalkbrood, an invasive honey bee mycosis. The most effective essential oils were Thymus herba-barona, Thymus capitatus and Cinnamomum zeylanicum, which showed values of minimum fungicidal concentration and minimum sporicidal concentration ranging from 200 to 400 ppm. Carvacrol was the main component of Thymus capitatus and Thymus herba-barona oils, whereas cinnamic aldehyde prevailed in Cinnamomum zeylanicum oil. Further in-apiary studies will allow the evaluation of side effects on bees and residues in hive products.

Honey bees increase social distancing when facing the ectoparasite Varroa destructor.

Social distancing in response to infectious diseases is a strategy exhibited by human and nonhuman animals to counteract the spread of pathogens and/or parasites. Honey bee (Apis mellifera) colonies are ideal models to study this behavior because of the compartmentalized structure of these societies, evolved under exposure to parasite pressure and the need to ensure efficient functioning. Here, by using a combination of spatial and behavioral approaches, we investigated whether the presence of the ectoparasite mite Varroa destructor induces changes in the social organization of A. mellifera colonies that could reduce the spread of the parasite. Our results demonstrated that honey bees react to the intrusion of V. destructor by modifying space use and social interactions to increase the social distancing between young (nurses) and old (foragers) cohorts of bees. These findings strongly suggest a behavioral strategy not previously reported in honey bees to limit the intracolony parasite transmission.

Twenty New Records of Bees (Hymenoptera, Apoidea) for Sardinia (Italy).

In Sardinia, the second largest Mediterranean island, 316 species of bees are known. Here, for the first time, the following 20 taxa are reported: Colletes cunicularius (Linnaeus, 1761), and C. eous Morice, 1904 (Colletidae); Andrena humilis Imhoff, 1832, A. granulosa Pérez, 1902, A. cineraria (Linnaeus, 1758), A. pallitarsis Pérez, 1903, A. rugulosa Stöckhert, 1935, A. savignyi Spinola, 1838, and A. tenuistriata Pérez, 1895 (Andrenidae); Sphecodes reticulatus Thomson, 1870 (Halictidae); Lithurgus tibialis Morawitz, 1875, Chelostoma emarginatum (Nylander, 1856), Dioxys cinctus (Jurine, 1807), Coelioxys caudatus Spinola, 1838, C. obtusus Pérez, 1884, and Megachile ericetorum (Lepeletier, 1841) (Megachilidae); and Nomada melathoracica Imhoff, 1834, N. pulchra Arnold, 1888, Eucera proxima Morawitz, 1875 and Tetralonia malvae (Rossi, 1790) (Apidae). N. pulchra is reported for the first time in Italy.

How the Infestation Level of Varroa destructor Affects the Distribution Pattern of Multi-Infested Cells in Worker Brood of Apis mellifera.

The mite Varroa destructor, the main ectoparasite of honey bees, is a threat to apiculture worldwide. Understanding the ecological interactions between Varroa and honeybees is fundamental for reducing mite impact in apiaries. This work assesses bee colonies with various Varroa infestation levels in apiaries to determine: (1) the relationship between multi-infested brood cells and brood infestation level, (2) the damage caused by Varroa to parasitized honey bee pupae, and (3) mite reproduction rate at various infestation levels. Data were collected from 19 worker brood combs, each from a different colony, ranging from 160 to 1725 (mean = 706) sealed cells per comb. Mite distribution was aggregated, ranging from about 2% to 74% infested cells per comb. The percentage of cells invaded by one, two, three, four, or more than four foundress mites, as a function of infestation level, was estimated by five highly significant (p < 0.0001) second-degree polynomial regression equations. The correction factors found could increase the precision of prediction models. Varroa fertility and adult bee longevity decreased as multi-infestation levels increased, and the implications of this relationship are discussed. Finally, these findings could improve sampling methods and the timing of mite treatments in apiaries, thus favoring sustainable management strategies.

Correction: Floris, I., et al. How the Infestation Level of Varroa destructor Affects the Distribution Pattern of Multi-Infested Cells in Worker Brood of Apis mellifera. Veterinary Science 2020, 7, 136.

We have recently been made aware by the reviewer and the Journal Editorial Offices of the following weaknesses in our recent paper [...].

Propolis Consumption Reduces Nosema ceranae Infection of European Honey Bees (Apis mellifera).

Nosema ceranae is a widespread obligate intracellular parasite of the ventriculus of many species of honey bee (Apis), including the Western honey bee Apis mellifera, in which it may lead to colony death. It can be controlled in A. mellifera by feeding the antibiotic fumagillin to a colony, though this product is toxic to humans and its use has now been banned in many countries, so in beekeeping, there exists a need for alternative and safe products effective against N. ceranae. Honeybees produce propolis from resinous substances collected from plants and use it to protect their nest from parasites and pathogens; propolis is thought to decrease the microbial load of the hive. We hypothesized that propolis might also reduce N. ceranae infection of individual bees and that they might consume propolis as a form of self-medication. To test these hypotheses, we evaluated the effects of an ethanolic extract of propolis administered orally on the longevity and spore load of experimentally N. ceranae-infected worker bees and also tested whether infected bees were more attracted to, and consumed a greater proportion of, a diet containing propolis in comparison to uninfected bees. Propolis extracts and ethanol (solvent control) increased the lifespan of N. ceranae-infected bees, but only propolis extract significantly reduced spore load. Our propolis extract primarily contained derivatives of caffeic acid, ferulic acid, ellagic acid and quercetin. Choice, scan sampling and food consumption tests did not reveal any preference of N. ceranae-infected bees for commercial candy containing propolis. Our research supports the hypothesis that propolis represents an effective and safe product to control N. ceranae but worker bees seem not to use it to self-medicate when infected with this pathogen.

Reversed-phase liquid chromatographic profile of free amino acids in strawberry-tree (Arbutus unedo L.) honey.

The typical profile of the free amino acids contained in strawberry-tree (Arbutus unedo L.) honey is reported for the first time. An optimized reversed-phase liquid chromatographic (RP-LC) method with phenyl isothiocyanate precolumn derivatization was used. Fourteen free amino acids were identified and quantified in 16 analytical samples. Proline (65.63%) was found to be the most abundant free amino acid, followed by glutamic acid (6.49%), arginine (5.21%), alanine (5.17%), and phenylalanine (4.97%). The total free amino acid content of strawberry-tree honey (average value, 436 mg/kg) was found to be low in comparison to amounts cited in the literature concerning unifloral honeys. The analytical method was optimized and fully validated in terms of detection and quantitation limits, precision (by testing repeatability and reproducibility), linearity, and bias (by means of recovery tests). The acceptability of the validation protocol results was verified using Horwitz's mathematical model and AOAC guidelines.

Susceptibility of Environmentally Friendly Sheep Wool Insulation Panels to the Common Clothes Moth Tineola bisselliella in Laboratory Assays.

In this study the resistance opposed to Tineola bisselliella larvae by a commercial sheep-wool panel incorporating borate salts was determined under laboratory conditions. The susceptibility of clothes moth larvae to different concentrations of disodium octaborate tetrahydrate (DOT) incorporated in pure wool was also determined. The commercial wool panel showed a remarkable resistance to moth attack compared with pure untreated wool, and the damage to panel samples was limited to their surface. As a result of bioassays exposing larvae to pure wool treated with DOT, a concentration dependent effect was observed, achieving a good efficacy at an application rate between 40-100 mg/mL. This study highlights the need to protect wool-based construction material with appropriate insecticidal (antifeedant or repellent) substances and supports the development of eco-sustainable solutions.