Exploring honeybee-mite immune interactions for developing RNAi based control technologies of Varroa destructor

The economic impact of beekeeping, including crop pollination, is very difficult to assess, but, according to the Italian Federation of Beekeepers (FAI), it can be estimated around 1,500 millions of euros annually, generated by about one million of hives. Honeybee colony losses have been a major problem since the beginning of modern apiculture; however, in 2006, the dramatic dimension of this phenomenon attracted the attention of the public opinion and the increasing interest of the scientific community. After a few years of intense investigation, a specific causal agent for the widespread colony losses was not found, but rather a multifactorial origin was proposed for this syndrome. It is now largely accepted that both landscape deterioration and agrochemicals can be directly or indirectly responsible for colony losses. However, several monitoring programs indicate that the large majority of losses are associated with the co-presence of the mite Varroa destructor and the Deformed Wing Virus (DWV). V. destructor is an ectoparasitic mite that creates a wound in the host’s cuticle through which it feeds on haemolymph and fat body, representing an important stress factor that weakens honeybee colonies and promotes the spreading of viral diseases. In order to facilitate feeding, V. destructor delivers a complex of factors, including proteins, through its salivary secretions. The scarcity of information about the sialome of the mite limits the functional analysis of the host regulation process and, thus, the opportunity to impair the mite’s fitness using biotechnological approaches. Here, we have used a functional genomics pipeline to identify V. destructor candidate salivary proteins, along with in situ hybridization detection to assess their expression in salivary glands. Using this approach, we identified a chitinase (Vd-CHI), specifically expressed in salivary glands, which affects mite survival and causes alterations of honeybee immune response. In particular, gene knockdown experiments revealed that Vd-CHI deficient mites tend to show a reduced survival as a likely consequence of reduced feeding capacity on honeybee pupae, due to the lack of chitinoloytic activity that favours the patency of the feeding wound. The importance of Vd-CHI for the feeding success of Varroa destructor is also supported by the upregulation of this gene during the reproductive phase of the mite, when the adult female creates large communal feeding site for her and the offspring, usually in the middle of the 2nd abdominal sternite of honeybee pupae. Vd-CHI has also an impact on honeybee’s immune response, determining the upregulation of an endogenous chitinase and the downregulation of an endocuticle structural glycoprotein, associated with wound healing and Varroa-resistance. On the other hand, infestation by Vd-CHI deficient mites led to upregulation of β-1,3-glucan binding protein and hymenoptaecin, as a likely consequence of bacterial infections, suggesting that Vd-CHI could also prevent detrimental bacterial proliferation. Collectively, these results shed light on Varroa-honeybee interaction, confirming that V. destructor sialome plays an important role in host exploitation. In particular, V. destructor feeding success on A. mellifera is mediated by the action of Vd-CHI, which probably facilitates the opening and patency of the feeding wound in the honeybee’s cuticle. Indeed, this chitinase is likely involved both in maintaining the feeding site open, by interfering with the regular healing process of cuticle, and in limiting opportunistic infections, by priming antimicrobial defenses. The present work contributes to a better knowledge of salivary repertoire of V. destructor and also demonstrates that dsRNA targeting of genes expressed in salivary glands can offer a promising new tool for controlling the mite

Ecological and Molecular Interactions between Insects and Fungi

Insects and fungi represent two of the most widespread groupings of organisms in nature, occurring in every kind of ecological context and impacting agriculture and other human activities in various ways. Moreover, they can be observed to reciprocally interact, establishing a wide range of symbiotic relationships, from mutualism to antagonism. The outcome of these relationships can in turn affect the extent at which species of both organisms can exert their noxious effects, as well as the management practices which are to be adopted to counter them. In conjunction with the launch of a Special Issue of Microorganisms with the same title, this article offers a general overview of the manifold aspects related to such interactions from the perspective of implementing our capacity to regulate them in a direction more favorable for the environment, crop production and human health

Anti-Insect Properties of Penicillium Secondary Metabolites

: In connection with their widespread occurrence in diverse environments and ecosystems, fungi in the genus Penicillium are commonly found in association with insects. In addition to some cases possibly implying a mutualistic relationship, this symbiotic interaction has mainly been investigated to verify the entomopathogenic potential in light of its possible exploitation in ecofriendly strategies for pest control. This perspective relies on the assumption that entomopathogenicity is often mediated by fungal products and that Penicillium species are renowned producers of bioactive secondary metabolites. Indeed, a remarkable number of new compounds have been identified and characterized from these fungi in past decades, the properties and possible applications of which in insect pest management are reviewed in this paper

Phylogenetic Characterization of Botryosphaeria Strains Associated with Asphondylia Galls on Species of Lamiaceae

In the last decade, Botryosphaeria dothidea has been steadily reported as an associate of gall midges (Diptera, Cecidomyiidae) in a variety of host plants and ecological settings. This cosmopolitan fungus is well-known for its ability to colonize many plant species, as both a pathogen and an endophyte. Thus, the shift from this general habit to a lifestyle involving a strict symbiotic relationship with an insect introduces expectancy for possible strain specialization which could reflect separated phylogenetic lineages. Considering the recent taxonomic revision concerning species of Botryosphaeria, we evaluated the phylogenetic relationships among strains recovered from Asphondylia galls collected on several species of Lamiaceae in Poland and in Italy, and all the currently accepted species in this genus. A number of strains previously characterized from gall samples from Australia and South Africa, whose genetic marker sequences are deposited in GenBank, were also included in the analysis. As a result, full identity as B. dothidea is confirmed for our isolates, while strains from the southern hemisphere grouped separately, indicating the existence of genetic variation related to the geographic origin in the association with gall midges

Neonicotinoid Clothianidin reduces honey bee immune response and contributes to Varroa mite proliferation

The neonicotinoid Clothianidin has a negative impact on NF-\u3baB signaling and on immune responses controlled by this transcription factor, which can boost the proliferation of honey bee parasites and pathogens. This effect has been well documented for the replication of deformed wing virus (DWV) induced by Clothianidin in honey bees bearing an asymptomatic infection. Here, we conduct infestation experiments of treated bees to show that the immune-suppression exerted by Clothianidin is associated with an enhanced fertility of the parasitic mite Varroa destructor, as a possible consequence of a higher feeding efficiency. A conceptual model is proposed to describe the synergistic interactions among different stress agents acting on honey bees

Neonicotinoid Clothianidin reduces honey bee immune response and contributes to Varroa mite proliferation

The neonicotinoid Clothianidin has a negative impact on NF-κB signaling and on immune responses controlled by this transcription factor, which can boost the proliferation of honey bee parasites and pathogens. This effect has been well documented for the replication of deformed wing virus (DWV) induced by Clothianidin in honey bees bearing an asymptomatic infection. Here, we conduct infestation experiments of treated bees to show that the immune-suppression exerted by Clothianidin is associated with an enhanced fertility of the parasitic mite Varroa destructor, as a possible consequence of a higher feeding efficiency. A conceptual model is proposed to describe the synergistic interactions among different stress agents acting on honey bees

A soil fungus confers plant resistance against a phytophagous insect by disrupting the symbiotic role of its gut microbiota

Plants generate energy flows through natural food webs, driven by competition for resources among organisms, which are part of a complex network of multitrophic interactions. Here, we demonstrate that the interaction between tomato plants and a phytophagous insect is driven by a hidden interplay between their respective microbiotas. Tomato plants colonized by the soil fungus Trichoderma afroharzianum, a beneficial microorganism widely used in agriculture as a biocontrol agent, negatively affects the development and survival of the lepidopteran pest Spodoptera littoralis by altering the larval gut microbiota and its nutritional support to the host. Indeed, experiments aimed to restore the functional microbial community in the gut allow a complete rescue. Our results shed light on a novel role played by a soil microorganism in the modulation of plant-insect interaction, setting the stage for a more comprehensive analysis of the impact that biocontrol agents may have on ecological sustainability of agricultural systems

Aspergillus-bees: A dynamic symbiotic association

Besides representing one of the most relevant threats of fungal origin to human and animal health, the genus Aspergillus includes opportunistic pathogens which may infect bees (Hymenoptera, Apoidea) in all developmental stages. At least 30 different species of Aspergillus have been isolated from managed and wild bees. Some efficient behavioral responses (e.g., diseased brood removal) exerted by bees negatively affect the chance to diagnose the pathology, and may contribute to the underestimation of aspergillosis importance in beekeeping. On the other hand, bee immune responses may be affected by biotic and abiotic stresses and suffer from the loose co-evolutionary relationships with Aspergillus pathogenic strains. However, if not pathogenic, these hive mycobiota components can prove to be beneficial to bees, by affecting the interaction with other pathogens and parasites and by detoxifying xenobiotics. The pathogenic aptitude of Aspergillus spp. likely derives from the combined action of toxins and hydrolytic enzymes, whose effects on bees have been largely overlooked until recently. Variation in the production of these virulence factors has been observed among strains, even belonging to the same species. Toxigenic and non-toxigenic strains/species may co-exist in a homeostatic equilibrium which is susceptible to be perturbed by several external factors, leading to mutualistic/antagonistic switch in the relationships between Aspergillus and bees

Cryptic Diversity in Cladosporium cladosporioides Resulting from Sequence-Based Species Delimitation Analyses

Cladosporium cladosporioides is an extremely widespread fungus involved in associations ranging from mutualistic to pathogenic and is the most frequently represented Cladosporium species in sequence databases, such as Genbank. The taxonomy of Cladosporium species, currently based on the integration of molecular data with morphological and cultural characters, is in frequent need of revision. Hence, the recently developed species delimitation methods can be helpful to explore cryptic diversity in this genus. Considering a previous study that reported several hypothetical species within C. cladosporioides, we tested four methods of species delimitation using the combined DNA barcodes internal transcribed spacers, translation elongation factor 1-α and actin 1. The analyses involved 105 isolates, revealing that currently available sequences of C. cladosporioides in GenBank actually represent more than one species. Moreover, we found that eight isolates from this set should be ascribed to Cladosporium anthropophilum. Our results revealed a certain degree of discordance among species delimitation methods, which can be efficiently treated using conservative approaches in order to minimize the risk of considering false positives