The use of honey bees (Apis mellifera L.) to monitor airborne particulate matter and assess health effects on pollinators.

The honey bee Apis mellifera has long been recognized as an ideal bioindicator for environmental pollution. These insects are exposed to pollutants during their foraging activities, making them effective samplers of environmental contaminants, including heavy metals, pesticides, radionuclides, and volatile organic compounds. Recently, it has been demonstrated that honey bees can be a valuable tool for monitoring and studying airborne PM pollution, a complex mixture of particles suspended in the air, known to have detrimental effects on human health. Airborne particles attached to the bees can be characterised for their morphology, size, and chemical composition using a scanning electron microscopy coupled with X-ray spectroscopy, thus providing key information on the emission sources of the particles, their environmental fate, and the potential to elicit inflammatory injury, oxidative damage, and other health effects in living organisms. Here, we present a comprehensive summary of the studies involving the use of honey bees to monitor airborne PM, including the limits of this approach and possible perspectives. The use of honey bees as a model organism for ecotoxicological studies involving pollutant PM is also presented and discussed, further highlighting the role of the bees as a cornerstone of human, animal, and environmental health, according to the principles of the "One Health" approach.

Distribution and prevalence of viral genomes in Italian populations of the invasive brown marmorated stink bug Halyomorpha halys.

Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species that causes significant agricultural losses, especially to orchard fruits, vegetables, herbaceous and ornamental plants. It is also a nuisance pest that seeks shelter in indoor spaces during the winter months. Harnessing the H. halys virome can result in new environmentally sustainable approaches to contain its populations and its relatated agricultural damages. In this study, RNA-Seq data were used to explore the virome associated to ten field populations collected in the Lombardy region in Northern Italy. We identified six complete viral genomes, three of which were previously unknown, belonging to the orders Reovirales, Articulavirales, Ghabrivirales, Durnavirales, and Picornavirales. The prevalence of the six viruses was evaluated by Real-time reverse transcription-quantitative PCR on eighty individuals. Halyomorpha halys ifla-like virus 2 turned out to be the most geographically widespread virus, as it was found in more than 50% of the analyzed insects and in nine out of the ten sampling locations. Moreover, in some individuals, this iflavirus was found in association with each of the other viruses in various combinations that involved up to four viruses. Further studies on such virus-virus interactions and their relationships with the insect host may open the possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of H. halys.

Origin of non-exhaust PM in cities by individual analysis of particles collected by honey bees (Apis mellifera).

Urban areas present multiple challenges to scientists interested in unraveling the source, transport, and fate of airborne particulate matter (PM). Airborne PM consists of a heterogeneous mixture of particles with different sizes, morphologies, and chemical compositions. However, standard air quality stations only detect the mass concentration of PM mixtures with aerodynamic diameters ≤10 µm (PM10) and/or ≤ 2.5 µm (PM2.5). During honey bee foraging flights, airborne PM up to 10 µm in size attaches to their bodies, making them suitable for collecting spatiotemporal data on airborne PM. The individual particulate chemistry of this PM can be assessed using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy on a sub-micrometer scale, allowing accurate identification and classification of the particles. Herein, we analyzed the PM fractions of 10-2.5 µm, 2.5-1 µm, and below 1 µm in average geometric diameter collected by bees from hives located in the city of Milan, Italy. Bees showed contamination by natural dust, originating from soil erosion and rock outcropping in the foraging area, and particles with recurrent heavy metal content, most likely attributed to vehicular braking systems and possibly tires (non-exhaust PM). Notably, approximately 80% of non-exhaust PM was ≤1 µm in size. This study provides a possible alternative strategy to apportion the finer fraction of PM in urban areas and determine citizens' exposure. Our findings may also prompt decision-makers to issue policy addressal for non-exhaust pollution, especially for the ongoing restructuring of European regulations on mobility and the shift toward electric vehicles whose contribution to PM pollution is debated.

Effects of oral exposure to brake wear particulate matter on the springtail Orthonychiurus folsomi.

Most of the heavy metals in urban environments derives from road traffic, particularly from tyres and brake wear (non-exhaust emission sources). These pollutants contaminate the soil, where several organisms have a primary ecosystem role (e.g., springtails, ants, earthworms). Springtails (Collembola) are soil-dwelling animals regulating soil fertility, flow of energy through above- and below-ground food webs, and they contribute to soil microbial community dispersion and biodiversity maintenance. In this study we investigated the ecotoxicological effects of oral exposure to particles emitted from brake pads and cast-iron brake discs in the euedaphic collembola species Orthonychiurus folsomi under laboratory conditions. Our results showed that chronic exposure to brake wear particles can have sub-lethal effects both at low and high concentrations and it can cause histological alterations. Here, SEM-EDX was applied to observe the particulate and we found its chemical markers in the gut and faeces of collembola, while histological analysis detected alterations of the digestive and reproductive systems and of the abdominal fat body at high concentrations.

Rethinking the Connections between Ecosystem Services, Pollinators, Pollution, and Health: Focus on Air Pollution and Its Impacts.

Ecosystems provide many services that are essential for human activities and for our well-being. Many regulation services are interconnected and are fundamental in mitigating and hindering the negative effects of several phenomena such as pollution. Pollution, in particular airborne particulate matter (PM), represents an important risk to human health. This perspective aims at providing a current framework that relates ecosystem services, regulating services, pollination, and human health, with particular regards to pollution and its impacts. A quantitative literature analysis on the topic has been adopted. The health repercussions of problems related to ecosystem services, with a focus on the effects of atmospheric particulate matter, have been highlighted in the work throughout a case study. In polluted environments, pollinators are severely exposed to airborne PM, which adheres to the insect body hairs and can be ingested through contaminated food resources, i.e., pollen and honey. This poses a serious risk for the health of pollinators with consequences on the pollination service and, ultimately, for human health.

The Honey Bee Apis mellifera: An Insect at the Interface between Human and Ecosystem Health.

The concept of ecosystem services is widely understood as the services and benefits thatecosystems provide to humans, and they have been categorised into provisioning, regulating, supporting, and cultural services. This article aims to provide an updated overview of the benefits that the honey bee Apis mellifera provides to humans as well as ecosystems. We revised the role of honey bees as pollinators in natural ecosystems to preserve and restore the local biodiversity of wild plants; in agro-ecosystems, this species is widely used to enhance crop yield and quality, meeting the increasing food demand. Beekeeping activity provides humans not only with high-quality food but also with substances used as raw materials and in pharmaceuticals, and in polluted areas, bees convey valuable information on the environmental presence of pollutants and their impact on human and ecosystem health. Finally, the role of the honey bee in symbolic tradition, mysticism, and the cultural values of the bee habitats are also presented. Overall, we suggest that the symbolic value of the honey bee is the most important role played by this insect species, as it may help revitalise and strengthen the intimate and reciprocal relationship between humans and the natural world, avoiding the inaccuracy of considering the ecosystems as mere providers of services to humans.

Influence of Microclimate Factors on Halyomorpha halys Dehydration.

Understanding the interaction between insects and microclimate can be essential in order to plan informed and efficient treatments against agricultural pests. Microclimatic factors such as humidity and temperature can influence the population dynamics of the invasive agricultural pest Halyomorpha halys, the brown marmorated stink bug. The aim of this work was to evaluate the level of transpiration of H. halys in dry, normal and humid microclimates according to the sex, physiological conditions and developmental stage of individuals. Water loss during diapause and the effect of population density on insects' transpiration were also assessed, as were the nutritional preferences of adults upon exiting diapause. Our data demonstrate that microclimatic conditions significantly influence the transpiration of this pest species. The effect of sex and feeding status on insects' water loss is marked, while population density does not influence water loss in diapausing individuals. The first nutritional need of the overwintering generations is represented by hydration, likely due to the water loss during diapause.

Disentangling multiple PM emission sources in the Po Valley (Italy) using honey bees.

Particulate matter (PM) is a complex mixture of airborne chemical compounds commonly classified by their aerodynamic diameter. Although PM toxicity strongly depends on the morphology, chemical composition, and dimensions of particles, exposure limits set by environmental organisations only refer to the mean mass concentration of PM sampled daily or annually by monitoring stations. In this study, we used honey bees as sensors of airborne PM10 and PM2.5 in a highly polluted area of the Po Valley, northern Italy. Honey bees are an efficient sampler of airborne PM because, during flight and foraging activities, their pubescence promotes the accumulation of electrical charge on the body surface owing to air resistance, thus enhancing airborne PM attraction. Particles attached to the body of bees are readily accessible for physico-chemical characterisation using a scanning electron microscope coupled with X-ray spectroscopy (SEM/EDX). Our results demonstrate that residents in the study area are intermittently but chronically exposed to a well-defined spectrum of metal-bearing particles and mineral phases known to induce specific health outcomes. The morphology, size, and chemical composition of PM10 and PM2.5 detected on bees in the monitoring area were indicative of traffic, agricultural operations, and high-temperature combustion processes. The contribution of the A1 Milano-Bologna highway, local wheat and alfalfa cultivation, and the Parma incineration plant were clearly distinguishable. Our data also demonstrated that PM exposure levels may vary sharply throughout the year based on recurrent local activities.

Vehicle-derived ultrafine particulate contaminating bees and bee products.

Despite adverse health effects, ultrafine particulate matter (UFP), i.e., PM less than 0.1 µm in diameter, is an emerging pollutant not subject to regulation. UFP may cause both lung inflammation and cardiopulmonary disease and may enter the brain directly via the olfactory bulb, affecting the nervous system. In highly urbanized environments, diesel and gasoline vehicles are among the major sources of UFP including combustion-generated solid particle pollutant and metal-based particles. Metal-based UFP are of much concern, as they may promote inflammation and DNA damage via oxidative stress with generation of free radicals and reactive oxygen species (ROS). We used the honeybee as an alternative sampling system of UFP in an area of the Po Valley (Northern Italy), which is subject to intense traffic. Worker bees are widely recognised as efficient samplers of air pollutants, including airborne PM. During flight and foraging activity, pubescence of the bees promotes the accumulation of electrical charge on the body's surface, enhancing attraction to air pollutants. Bees living near the main Italian highway, the Autostrada A1, displayed a contamination of nanosized Fe-oxides/hydroxides and baryte. Sources of Fe-bearing and baryte ultrafine particles are primarily the vehicles speeding on the motorway. Pollen collected by forager bees and honey produced by the bee colony displayed contamination by nanosized Fe-oxides/hydroxides and baryte. Such a contamination exposes pollinators and humans to UFP ingestion, endangering the safety of food produced at traffic-influenced sites. Given the global spread of traffic, our findings suggest that exposure and environmental impact of ultrafine Fe-oxides/hydroxides and baryte are potentially ubiquitous, although usually overlooked in environmental policy discussions.

Cannibalism in the Brown Marmorated Stink Bug Halyomorpha halys (Stål).

The phytophagous brown marmorated stink bug Halyomorpha halys (Stål) is known to exhibit cannibalistic behaviour towards eggs. Here, we provide evidence of cannibalism among overwintering H. halys adults. Since diapausing individuals have high physiological demands for surviving long periods under stressful conditions, including the risk of depletion of metabolic reserves and desiccation, we assumed that nutritional and water requirements can be met by intraspecific predation. The role of aggregative behaviour in promoting cannibalism is also discussed. Given its evolutionary advantage, this trait should be maintained over generations and may be more widespread than previously considered in species that display aggregative behaviour during adverse seasons.

Particulate matter collection by honey bees (Apis mellifera, L.) near to a cement factory in Italy.

Industrial activities play a key role in the economic well-being of a country but they usually involve processes with a more or less profound environmental impact, including emission of pollutants. Among them, much attention has been given to airborne particulate matter (PM) whose exposure is ubiquitous and linked with several adverse health effects mainly due to its size and chemical composition. Therefore, there is a strong need to exploit monitoring systems for airborne PM able to provide accurate information on the potential health hazards and the specific emission sources for the implementation of adequate control strategies. The honey bee (Apis mellifera, L.) is widely used as an indicator of environmental pollution: this social hymenopteran strongly interacts with vegetables, air, soil, and water surrounding the hive and, as a consequence, pollutants from these sources are translated to the insect and to the hive products. During the wide-ranging foraging activity, the forager bee is known to collect samples of the main airborne PM pollutants emitted from different sources and therefore it can be used as an efficient PM sampler. In the present research, PM contaminating forager bees living nearby a cement factory and several kilometers away from it has been analysed and characterised morphologically, dimensionally and chemically through SEM/EDX. This provided detailed information on the role of both the cement manufacturing activities and the vehicular traffic as sources of airborne PM. This may indeed help the implementation of appropriate preventive and corrective actions that would effectively minimize the environmental spread of pollutant PM not only in areas close to the plant, but also in more distant areas.

Plant-mediated interspecific horizontal transmission of an intracellular symbiont in insects.

Intracellular reproductive manipulators, such as Candidatus Cardinium and Wolbachia are vertically transmitted to progeny but rarely show co-speciation with the host. In sap-feeding insects, plant tissues have been proposed as alternative horizontal routes of interspecific transmission, but experimental evidence is limited. Here we report results from experiments that show that Cardinium is horizontally transmitted between different phloem sap-feeding insect species through plants. Quantitative PCR and in situ hybridization experiments indicated that the leafhopper Scaphoideus titanus releases Cardinium from its salivary glands during feeding on both artificial media and grapevine leaves. Successional time-course feeding experiments with S. titanus initially fed sugar solutions or small areas of grapevine leaves followed by feeding by the phytoplasma vector Macrosteles quadripunctulatus or the grapevine feeder Empoasca vitis revealed that the symbionts were transmitted to both species. Explaining interspecific horizontal transmission through plants improves our understanding of how symbionts spread, their lifestyle and the symbiont-host intermixed evolutionary pattern.

Honey Bees (Apis mellifera, L.) as Active Samplers of Airborne Particulate Matter.

Honey bees (Apis mellifera L.) are bioindicators of environmental pollution levels. During their wide-ranging foraging activity, these hymenopterans are exposed to pollutants, thus becoming a useful tool to trace the environmental contaminants as heavy metals, pesticides, radionuclides and volatile organic compounds. In the present work we demonstrate that bees can also be used as active samplers of airborne particulate matter. Worker bees were collected from hives located in a polluted postmining area in South West Sardinia (Italy) that is also exposed to dust emissions from industrial plants. The area is included in an official list of sites of national interest for environmental remediation, and has been characterized for the effects of pollutants on the health of the resident population. The head, wings, hind legs and alimentary canal of the bees were investigated with Scanning Electron Microscopy coupled with X-ray spectroscopy (SEM-EDX). The analyses pointed to specific morphological and chemical features of the particulate, and resulted into the identification of three categories of particles: industry-, postmining-, and soil-derived. With the exception of the gut, all the analyzed body districts displayed inorganic particles, mostly concentrated in specific areas of the body (i.e. along the costal margin of the fore wings, the medial plane of the head, and the inner surface of the hind legs). The role of both past mining activities and the industrial activity close to the study area as sources of the particulate matter is also discussed. We conclude that honey bees are able to collect samples of the main airborne particles emitted from different sources, therefore could be an ideal tool for monitoring such a kind of pollutants.

Wolbachia is not all about sex: male-feminizing Wolbachia alters the leafhopper Zyginidia pullula transcriptome in a mainly sex-independent manner.

Wolbachia causes the feminization of chromosomally male embryos in several species of crustaceans and insects, including the leafhopper Zyginidia pullula. In contrast to the relatively well-established ecological aspects of male feminization (e.g., sex ratio distortion and its consequences), the underlying molecular mechanisms remain understudied and unclear. We embarked on an exploratory study to investigate the extent and nature of Wolbachia's effect on gene expression pattern in Z. pullula. We sequenced whole transcriptomes from Wolbachia-infected and uninfected adults. 18147 loci were assembled de novo, including homologs of several Drosophila sex determination genes. A number of transcripts were flagged as candidate Wolbachia sequences. Despite the resemblance of Wolbachia-infected chromosomal males to uninfected and infected chromosomal females in terms of sexual morphology and behavior, principal component analysis revealed that gene expression patterns did not follow these sexual phenotype categories. The principal components generated by differentially expressed genes specified a strong sex-independent Wolbachia effect, followed by a weaker Wolbachia-sexual karyotype interaction effect. Approaches to further examine the molecular mechanism of Wolbachia-host interactions have been suggested based on the presented findings.

Bacterial endosymbiont localization in Hyalesthes obsoletus, the insect vector of Bois noir in Vitis vinifera.

One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by "Candidatus Phytoplasma solani" and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the "Candidatus Sulcia muelleri" cluster in the Bacteroidetes and in the "Candidatus Purcelliella pentastirinorum" group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name "Candidatus Vidania fulgoroideae." Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and "Candidatus Cardinium hertigii." Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As "Ca. Sulcia" is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between "Ca. Sulcia" and "Ca. Purcelliella," "Ca. Vidania," or both.

Wolbachia as an "infectious" extrinsic factor manipulating host signaling pathways.

Wolbachia pipientis is a widespread endosymbiont of filarial nematodes and arthropods. While in worms the symbiosis is obligate, in arthropods Wolbachia induces several reproductive manipulations (i.e., cytoplasmic incompatibility, parthenogenesis, feminization of genetic males, and male-killing) in order to increase the number of infected females. These various phenotypic effects may be linked to differences in host physiology, and in particular to endocrine-related processes governing growth, development, and reproduction. Indeed, a number of evidences links Wolbachia symbiosis to insulin and ecdysteroid signaling, two multilayered pathways known to work antagonistically, jointly or even independently for the regulation of different molecular networks. At present it is not clear whether Wolbachia manipulates one pathway, thus affecting other related metabolic networks, or if it targets both pathways, even interacting at several points in each of them. Interestingly, in view of the interplay between hormone signaling and epigenetic machinery, a direct influence of the "infection" on hormonal signaling involving ecdysteroids might be achievable through the manipulation of the host's epigenetic pathways.

Bacteriocyte-like cells harbour Wolbachia in the ovary of Drosophila melanogaster (Insecta, Diptera) and Zyginidia pullula (Insecta, Hemiptera).

Wolbachia is the most widespread bacterial endosymbiont in insects. It is responsible for a variety of reproductive alterations of the hosts. Wolbachia is transmitted through the germline from mother to offspring and, in rare cases, between individuals. This implies that acquired properties (through symbiosis with Wolbachia) can become heritable. We investigated the transovarial inheritance of Wolbachia in two phylogenetically distant insects, Drosophila melanogaster and Zyginidia pullula. We detected in both systems bacteriocyte-like cells, densely packed with Wolbachia endosymbionts, at the tip of the ovarioles. Bacteriocytes are cells specialized to harbour bacteria, typical of mutualistic insect symbiosis. Our observations of bacteriocyte-like cells harbouring Wolbachia in the ovary emphasize the plasticity of the female reproductive system of insects, which maintains its function while some cells are densely colonized by bacteria. In summary, there is evidence from different insects that bacteria which behave as parasites of reproduction are harboured by cells resembling bacteriocytes, which appear to mediate transmission of the bacteria to the progeny. It seems a valid hypothesis that the bacteriocyte-like cells that we observed are not the result of a co-evolution of host and symbiont, considering that Wolbachia is not an obligatory symbiont in Drosophila and Zyginidia.

Sex and stripping: The key to the intimate relationship between Wolbachia and host?

Wolbachia pipientis is known to infect only arthropods and nematodes (mainly filarial worms). A unique feature shared by the two Phyla is the ability to replace the exoskeleton, a process known as ecdysis. This shared characteristic is thought to reflect a common ancestry. Arthropod moulting is induced by the steroid hormone 20-hydroxyecdysone (20E) and a role for ecdysteroids in nematode ecdysis has also been suggested. Removing Wolbachia from filarial worms impairs the host's development. From analyses of the genome of Wolbachia harbored by the filarial nematode Brugia malayi and that of its host, the bacterium may provide a source of heme, an essential component of cytochrome P450's that are necessary for steroid hormone biosynthetic pathways.In arthropods, Wolbachia is a reproductive manipulator, inducing various phenotypic effects that may be due to differences in host physiology, in particular, endocrine-related processes governing development and reproduction. Insect steroids have well-defined roles in the coordination of multiple developmental processes, and in adults they control important aspects of reproduction, including ovarian development, oogenesis, sexual behavior, and in some taxa vitellogenin biosynthesis.According to some authors ecdysteroids may also act as sex hormones. In insects sex differentiation is generally thought to be a strictly genetic process, in which each cell decides its own sexual fate based on its sex chromosome constitution, but, surprisingly, recent data demonstrate that in Drosophila sex determination is not cell-autonomous, as it happens in mammals. Thus the presence of signals coordinating the development of a gender-specific phenotype cannot be excluded.This could explain why Wolbachia interferes with insect reproduction; and also could explain why Wolbachia interferes with insect development.Thus, is "sex (=reproduction) and stripping (=ecdysis)" the key to the intimate relationship between Wolbachia and its host?