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.

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.

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.

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.

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.

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.

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 [...].

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.

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.

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.

Inhibition of Paenibacillus larvae by an extracellular protein fraction from a honeybee-borne Brevibacillus laterosporus strain.

The inhibitory action that a Brevibacillus laterosporus strain isolated from the honeybee body causes against the American Foulbrood (AFB) etiological agent Paenibacillus larvae was studied by in-vitro experiments. A protein fraction isolated from B. laterosporus culture supernatant was involved in the observed inhibition of P. larvae vegetative growth and spore germination. As a result of LC-MS/MS proteomic analyses, the bacteriocin laterosporulin was found to be the major component of this fraction, followed by other antimicrobial proteins and substances including lectins, chaperonins, various enzymes and a number of putative uncharacterized proteins. The results obtained in this study highlight the potential of B. laterosporus as a biological control agent for preserving and improving honeybee health.

Resin foraging dynamics in Varroa destructor-infested hives: a case of medication of kin?

Social insects have evolved colony behavioral, physiological, and organizational adaptations (social immunity) to reduce the risks of parasitization and/or disease transmission. The collection of resin from various plants and its use in the hive as propolis is a clear example of behavioral defense. For Apis mellifera, an increased propolis content in the hive may correspond to variations in the microbial load of the colony and to a downregulation of an individual bee's immune response. However, many aspects of such antimicrobial mechanism still need to be clarified. Assuming that bacterial and fungal infection mechanisms differ from the action of a parasite, we studied the resin collection dynamics in Varroa destructor-infested honeybee colonies. Comparative experiments involving hives with different mite infestation levels were conducted in order to assess the amount of resin collected and propolis quality within the hive, over a 2-year period (2014 and 2015). Our study demonstrates that when A. mellifera colonies are under stress because of Varroa infestation, an increase in the number of resin foragers is recorded, even if a general intensification of the foraging activity is not observed. A reduction in the total polyphenolic content in propolis produced in infested versus uninfested hives was also noticed. Considering that different propolis types show varying levels of inhibition against a variety of honey bee pathogens in vitro, it would be very important to study the effects against Varroa of two diverse types of propolis: from Varroa-free and from Varroa-infested hives.

Feeding strategies and intraspecific competition in German yellowjacket (Vespula germanica).

The German yellowjacket (Vespula germanica) is an opportunist predator and a scavenger, whose eclectic diet also includes honey, brood, dead and live honey-bees. There is no evidence in this species of coordinated attacks against bees involving other conspecifics, although intraspecific competition has been already reported between two or more individuals during feeding. Our aim was to gain further knowledge on the feeding behavior of V. germanica in order to evaluate its role in an apiary. Sight observations of predation and necrophagy behaviors were carried out at the ground level near hives. We also investigated how intraspecific competition can influence the feeding display in this species. Our results confirm the major role of the German yellowjacket as a scavenger, because its diet is based mostly on bee carrions. Intraspecific competition during feeding was sometimes observed. When these events occurred, the interference of another wasp led to the bee escaping only in three cases. Our study also revealed that intraspecific competition events increase when the resource is fresh (predation vs necrophagy), and that the number of competing wasps was significantly higher when the food consisted of pupae and drones, compared to adult bees. When competition involved two individuals (the most frequent case), the winner was frequently the first wasp to reach the resource in both predation and necrophagy events. This suggests that the energy invested in foraging or predating activity and in defence of prey is usually rewarded.

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.

Agonistic interactions between the honeybee (Apis mellifera ligustica) and the European wasp (Vespula germanica) reveal context-dependent defense strategies.

Predator-prey relationships between sympatric species allow the evolution of defense behaviors, such as honeybee colonies defending their nests against predatory wasps. We investigated the predator-prey relationship between the honeybee (Apis mellifera ligustica) and the European wasp (Vespula germanica) by evaluating the effectiveness of attack and defense behaviors, which have coevolved in these sympatric species, as well as the actual damage and disturbance caused to the colonies under attack. Attack and defense behaviors were recorded in front of the hive to observe attacks at the hive entrance (68 attacks in 279 h) and at ground level on isolated and weakened honeybees close to the hive (465 attacks in 32 h). We found that V. germanica attacked the hive entrance infrequently due to the low success rate of this strategy and instead preferred a specialized attack method targeting adult honeybees at ground level, demonstrating opportunistic scavenger behavior. Individual honeybees usually responded effectively to an attack by recruiting an average of two nestmates, causing the wasp to flee, whereas collective balling behavior was only observed on four occasions. V. germanica does not appear to disrupt the foraging activity of the colonies under attack. We found that agonistic events supported by other nestmates were typically the most intense ones, involving physical combat and prolonged attacks at the entrance to the hive. These observations support the hypothesis that A. mellifera ligustica can adapt its behavior to match the severity of the threat and the context of the attack.

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.

Combination of beehive matrices analysis and ant biodiversity to study heavy metal pollution impact in a post-mining area (Sardinia, Italy).

Mining activities represent a major source of environment contamination. The aim of this study was to evaluate the use of bees and ants as bioindicators to detect the heavy metal impact in post-mining areas. A biomonitoring programme involving a combination of honeybee hive matrices analysis and ant biodiversity survey was conducted over a 3-year period. The experimental design involved three monitoring stations where repeated sampling activities focused on chemical detection of cadmium (Cd), chrome (Cr) and lead (Pb) from different matrices, both from hosted beehives (foraging bees, honey and pollen) and from the surrounding environment (stream water and soil). At the same time, ant biodiversity (number and abundance of species) was determined through a monitoring programme based on the use of pitfall traps placed in different habitats inside each mining site. The heavy metal content detected in stream water from the control station was always below the analytical limit of quantification. In the case of soil, the content of Cd and Pb from the control was lower than that of mining sites. The mean heavy metal concentrations in beehive matrices from mining sites were mainly higher than the control, and as a result of regression and discriminant analysis, forager bee sampling was an efficient environmental pollution bioindicator. Ant collection and identification highlighted a wide species variety with differences among habitats mostly associated with vegetation features. A lower variability was observed in the polluted landfill characterised by lack of vegetation. Combined biomonitoring with forager bees and ants represents a reliable tool for heavy metal environmental impact studies.

New experimental data on use of rotenone as an acaricide for control of Varroa destructor in honey bee colonies.

Three slow release experimental rotenone formulations were tested to evaluate their effectiveness against Varroa destructor Anderson & Trueman in colonies with sealed brood and to determine whether they left residues in honey and bees wax: we evaluated cardboard strip containing 1 g rotenone and two types of polyvinyl chloride (PVC) strips containing 1 (high-dose) and 0.5 (low-dose) g of rotenone, respectively. In general, the efficacy of the treatments, expressed as percentage of mite mortality, was highly variable in all treatment groups (range, 0-96.8%). The highest effectiveness was obtained with the high-dose-PVC strips, which caused an average percentage of mortality ranging between 47 and 69% in the adult bees and sealed brood, respectively. At the end of the treatment, rotenone residues ranged between 0.03 and 0.06 and 1.5-144.0 mg/kg in honey and wax, respectively. Rotenone residues in wax were still detectable 4 mo after the treatment period, whereas no residues were found in honey. The higher residues content and persistence recorded in wax samples, was probably due to the lipophilic nature of rotenone. A reduction in the amount of adults was recorded for the group treated with high-dose-PVC strips compared with the untreated colonies. Toxicological risks connected with the use of rotenone and the low maximum level recently fixed by European legislation (0.01 mg/kg) suggest that rotenone is not a good candidate for reducing varroa populations in honey bee colonies.