Entomophthoralean fungi overwinter with the bird cherry-oat aphid on bird cherry trees.

In Scandinavia, the bird cherry-oat aphid Rhopalosiphum padi overwinter as eggs on the bird cherry tree Prunus padus. Branches of P. padus were collected at the late February / early March from 17 locations in Norway over a three-year period. We found 3599 overwintering aphid eggs, 59.5% of which were dead. Further, a total of 879 overwintering fungus-killed cadavers were observed. These cadavers were found close to bud axils, where overwintering eggs were also usually attached. Cadavers were infected with either Zoophthora cf. aphidis or Entomophthora planchoniana. All the fungal-killed cadavers were filled with overwintering structures of Z. cf. aphidis (as resting spores) or E. planchoniana (as modified hyphal bodies). We found a significant negative correlation between eggs and cadavers per branch. However, both numbers of eggs and cadavers varied greatly between years and among tree locations. This is the first report of E. planchoniana overwintering in R. padi cadavers as modified hyphal bodies. We discuss whether P. padus may act as an inoculum reservoir for fungi infecting aphids in cereals in spring.

Strongwellsea selandia and Strongwellsea gefion (Entomophthorales: Entomophthoraceae), two new species infecting adult flies from genus Helina (Diptera: Muscidae).

Two new species from the genus Strongwellsea (Entomophthorales: Entomophthoraceae) that infect adult flies from the genus Helina (Muscidae) are described: Strongwellsea selandia Eilenberg & Humber infecting adult Helina evecta (Harris), and Strongwellsea gefion Eilenberg & Humber infecting adult Helina reversio (Harris). The descriptions are based on pathobiological, phenotypical and genotypical characters. The new species differ from other described members from the genus Strongwellsea by a) pathobiology as revealed by natural host species, b) morphology of primary conidia, c) color of resting spores, and d) genotypical clustering based on analysis of ITS2. The two new species have only been documented from North Zealand, Denmark.

Strongwellsea crypta (Entomophthorales: Entomophthoraceae), a new species infecting Botanophila fugax (Diptera: Anthomyiidae).

A new species from the genus Strongwellsea (Entomophthorales: Entomophthoraceae) is described: Strongwellsea crypta Eilenberg & Humber from adult Botanophila fugax (Meigen) (Diptera: Anthomyiidae). The description is based on pathobiological, phenotypical and genotypical characters. The abdominal holes in infected hosts develop rapidly and become strikingly large and edgy, almost rhomboid in shape. The new species S. crypta differs from S. castrans, the only described species infecting flies from Anthomyiidae, by: (a) naturally infecting another host species, (b) by having significantly longer primary conidia, and (c) by genotypical clustering separately from that species when sequencing ITS2.

Pathogenicity against hemipteran vector insects of a novel insect pathogenic fungus from Entomophthorales (Pandora sp. nov. inedit.) with potential for biological control.

A new but still unpublished entomopathogenic fungus (ARSEF13372) in the genus Pandora (Entomophthorales: Entomophthoraceae) was originally isolated from Cacopsylla sp. (Hemiptera: Psyllidae). Several species of the genus Cacopsylla vector phloem-borne bacteria of the genus 'Candidatus Phytoplasma', which cause diseases in fruit crops such as apple proliferation, pear decline and European stone fruit yellows. To determine Pandora's host range and biocontrol potential we conducted laboratory infection bioassays; Hemipteran phloem-feeding insects were exposed to conidia actively discharged from in vitro produced mycelial mats of standardized area. We documented the pathogenicity of Pandora sp. nov. to species of the insect families Psyllidae and Triozidae, namely Cacopsyllapyri L., C.pyricola (Foerster), C.picta (Foerster, 1848), C.pruni (Scopoli), C.peregrina (Foerster), and Trioza apicalis Foerster. The occurrence of postmortem signs of infection on cadavers within 10 days post inoculation proved that Pandora sp. nov. was infective to the tested insect species under laboratory conditions and significantly reduced mean survival time for C.pyri (summer form and nymph), C.pyricola, C.picta, C.pruni, C.peregrina and T.apicalis. Assessing a potential interaction between phytoplasma, fungus and insect host revealed that phytoplasma infection ('Candidatus Phytoplasma mali') of the vector C.picta and/or its host plant apple Malus domestica Borkh. did not significantly impact the survival of C.picta after Pandora sp. nov. infection. The results from infection bioassays were discussed in relation to Pandora sp. nov. host range and its suitability as biocontrol agent in integrated pest management strategies of psyllid pests, including vector species, in orchards.

Strong host specialization in fungus genus Strongwellsea (Entomophthorales).

The genus Strongwellsea (Entomophthorales) has a unique pathobiology. Infected adult dipteran hosts develop a large hole in their abdomens, through which conidia are actively discharged while the hosts are still alive. We analyzed the IST II region of Strongwellsea from 29 infected hosts representing 15 dipteran species from Anthomyiidae, Fanniidae, Muscidae, and Scathophagidae. Each genotype was found on only one host species or a few closely related host species. Strongwellsea genotypes infecting flies from Anthomyiidae represented a monophyletic lineage, including the species Strongwellsea castrans, while genotypes infecting Muscidae were very diverse and clustered at different places. All three host species from Fanniidae were infected with the same Strongwellsea genotype, namely the species Strongwellsea magna. It appears that members of the genus Strongwellsea are strongly adapted to their host species and have co-evolved.

Pandora formicae, a specialist ant pathogenic fungus: New insights into biology and taxonomy.

Among fungi from the order Entomophthorales (Entomophthoromycota), there are many specialized, obligatory insect-killing pathogens. Pandora formicae (Humber & Balazy) Humber is a rare example of an entomophthoralean fungus adapted to exclusively infect social insects: wood ants from the genus Formica. There is limited information available on P. formicae; many important aspects of this host-pathogen system remain hitherto unknown, and the taxonomical status of the fungus is unclear. Our study fills out some main gaps in the life history of P. formicae, such as seasonal prevalence and overwintering strategy. Field studies of infection prevalence show a disease peak in late summer and early autumn. Typical thick-walled entomophthoralean resting spores of P. formicae are documented and described for the first time. The proportion of cadavers with resting spores increased from late summer throughout autumn, suggesting that these spores are the main overwintering fungal structures. In addition, the phylogenetic status of Pandora formicae is outlined. Finally, we review the available taxonomical literature and conclude that the name P. formicae should be used rather than the name P. myrmecophaga for ant-infecting fungi displaying described morphological features.

Phylogenetic placement of two species known only from resting spores: Zoophthora independentia sp. nov. and Z. porteri comb nov. (Entomophthorales: Entomophthoraceae).

Molecular methods were used to determine the generic placement of two species of Entomophthorales known only from resting spores. Historically, these species would belong in the form-genus Tarichium, but this classification provides no information about phylogenetic relationships. Using DNA from resting spores, Zoophthora independentia, infecting Tipula (Lunatipula) submaculata in New York State, is now described as a new species and Tarichium porteri, described in 1942, which infects Tipula (Triplicitipula) colei in Tennessee, is transferred to the genus Zoophthora. We have shown that use of molecular methods can assist with determination of the phylogenetic relations of specimens within the form-genus Tarichium for an already described species and a new species for which only resting spores are available.

Standard methods for fungal brood disease research.

Chalkbrood and stonebrood are two fungal diseases associated with honey bee brood. Chalkbrood, caused by Ascosphaera apis, is a common and widespread disease that can result in severe reduction of emerging worker bees and thus overall colony productivity. Stonebrood is caused by Aspergillus spp. that are rarely observed, so the impact on colony health is not very well understood. A major concern with the presence of Aspergillus in honey bees is the production of airborne conidia, which can lead to allergic bronchopulmonary aspergillosis, pulmonary aspergilloma, or even invasive aspergillosis in lung tissues upon inhalation by humans. In the current chapter we describe the honey bee disease symptoms of these fungal pathogens. In addition, we provide research methodologies and protocols for isolating and culturing, in vivo and in vitro assays that are commonly used to study these host pathogen interactions. We give guidelines on the preferred methods used in current research and the application of molecular techniques. We have added photographs, drawings and illustrations to assist bee-extension personnel and bee scientists in the control of these two diseases.

A New Approach for Detecting Sublethal Effects of Neonicotinoids on Bumblebees Using Optical Sensor Technology.

Among insects, bees are important pollinators, providing many vital ecosystem services. The recent pollinator decline is threatening both their diversity and abundance. One of the main drivers of this decline is the extensive use of pesticides. Neonicotinoids, one of the most popular groups of pesticides, can be toxic to bees. In fact, numerous studies have found that neonicotinoids can cause sublethal effects, which can impair the biology, physiology, and colony survival of the bees. Yet, there are still knowledge gaps, and more research is needed to better understand the interaction between neonicotinoids and bees, especially in the field. A new optical sensor, which can automatically identify flying insects using machine learning, has been created to continuously monitor insect activity in the field. This study investigated the potential use of this sensor as a tool for monitoring the sublethal effects of pesticides on bumblebees. Bombus terrestris workers were orally exposed to field-realistic doses of imidacloprid. Two types of exposures were tested: acute and chronic. The flight activity of pesticide-exposed and non-exposed bumblebees was recorded, and the events of the insect flights recorded by the sensor were used in two ways: to extract the values of the wingbeat frequency and to train machine learning models. The results showed that the trained model was able to recognize differences between the events created by pesticide-exposed bumblebees and the control bumblebees. This study demonstrates the possibility of the optical sensor for use as a tool to monitor bees that have been exposed to sublethal doses of pesticides. The optical sensor can provide data that could be helpful in managing and, ideally, mitigating the decline of pollinators from one of their most major threats, pesticides.

The immunostimulatory role of an Enterococcus-dominated gut microbiota in host protection against bacterial and fungal pathogens in Galleria mellonella larvae.

Understanding the intricate interplay between the gut microbiota and the immune response in insects is crucial, given its diverse impact on the pathogenesis of various microbial species. The microbiota's modulation of the host immune system is one such mechanism, although its complete impact on immune responses remains elusive. This study investigated the tripartite interaction between the gut microbiota, pathogens, and the host's response in Galleria mellonella larvae reared under axenic (sterile) and conventional (non-sterile) conditions. The influence of the microbiota on host fitness during infections was evaluated via two different routes: oral infection induced by Bacillus thuringiensis subsp. galleriae (Btg), and topical infection induced by Metarhizium robertsii (Mr). We observed that larvae without a microbiota can successfully fulfill their life cycle, albeit with more variation in their developmental time. We subsequently performed survival assays on final-instar larvae, using the median lethal dose (LD50) of Btg and Mr. Our findings indicated that axenic larvae were more vulnerable to an oral infection of Btg; specifically, a dose that was calculated to be half-lethal for the conventional group resulted in a 90%-100% mortality rate in the axenic group. Through a dual-analysis experimental design, we could identify the status of the gut microbiota using 16S rRNA sequencing and assess the level of immune-related gene expression in the same group of larvae at basal conditions and during infection. This analysis revealed that the microbiota of our conventionally reared population was dominated entirely by four Enterococcus species, and these species potentially stimulated the immune response in the gut, due to the increased basal expression of two antimicrobial peptides (AMPs)-gallerimycin and gloverin-in the conventional larvae compared with the axenic larvae. Furthermore, Enterococcus mundtii, isolated from the gut of conventional larvae, showed inhibition activity against Btg in vitro. Lastly, other immune effectors, namely, phenoloxidase activity in the hemolymph and total reactive oxygen/nitrogen species (ROS/RNS) in the gut, were tested to further investigate the extent of the stimulation of the microbiota on the immune response. These findings highlight the immune-modulatory role of the Enterococcus-dominated gut microbiota, an increasingly reported microbiota assemblage of laboratory populations of Lepidoptera, and its influence on the host's response to oral and topical infections.

Ascosphaera subglobosa, a new spore cyst fungus from North America associated with the solitary bee Megachile rotundata.

Ascosphaera subglobosa (Eurotiomycetes: Onygenales) is newly described from the pollen provisions and nesting material of the solitary leaf-cutting bee Megachile rotundata in Canada and the western United States. This new species, related to A. atra and A. duoformis, is distinguished from other Ascosphaera species by its globose to subglobose ascospores, evanescent spore balls and unique nuclear ribosomal DNA sequences (ITS and LSU).

Effect of Probiotics on Tenebrio molitor Larval Development and Resistance against the Fungal Pathogen Metarhizium brunneum.

In recent years, the yellow mealworm (Tenebrio molitor L.) has demonstrated its potential as a mass-produced edible insect for food and feed. However, challenges brought on by pathogens in intensive production systems are unavoidable and require the development of new solutions. One potential solution is the supplementation of probiotics in the insect's diet to obtain the double benefits of improved growth and enhanced immune response. The aim of this study was to evaluate the effects of diet-based probiotic supplementation on T. molitor larval survival, growth, and resistance against a fungal pathogen. Three probiotic strains, namely Pediococcus pentosacceus KVL-B19-01 isolated from T. molitor and two commercialized strains for traditional livestock, Enterococcus faecium 669 and Bacillus subtilis 597, were tested. Additionally, when larvae were 9 weeks old, a pathogen challenge experiment was conducted with the fungus Metarhizium brunneum. Results showed that both P. pentosaceus and E. faecium improved larval growth and larval survival following fungal exposure compared to the non-supplemented control diet. Since B. subtilis did not improve larval performance in terms of either development or protection against M. brunneum, this study suggests the need for further research and evaluation of probiotic strains and their modes of action when considered as a supplement in T. molitor's diet.

Uptake and Survival of African Swine Fever Virus in Mealworm (Tenebrio molitor) and Black Soldier Fly (Hermetia illucens) Larvae.

Insect production offers a sustainable source of nutrients for livestock. This comes with a risk for transmission of pathogens from the insects into the livestock sector, including viruses causing serious diseases, such as African swine fever virus (ASFV), classical swine fever virus and foot-and-mouth disease virus. ASFV is known to survive for a long time within animal meat and byproducts. Therefore, we conducted experimental exposure studies of insects to ASFV using larvae of two key insect species produced for food and feed, the mealworm; Tenebrio molitor, and the black soldier fly, Hermetia illucens. The larvae were exposed to ASFV POL/2015/Podlaskie, via oral uptake of serum or spleen material from ASFV-infected pigs. Using qPCR, the amounts of viral DNA present immediately after exposure varied from ~104.7 to 107.2 genome copies per insect. ASFV DNA was detectable in the larvae of H. illucens for up to 3 days post exposure and in T. molitor larvae for up to 9 days post exposure. To assess the presence of infectious virus within the larvae and with this, the risk of virus transmission via oral consumption, pigs were fed cakes containing larvae exposed to ASFV. Pigs that consumed 50 T. molitor or 50 H. illucens virus-exposed larvae did not become infected with ASFV. Thus, it appears, that in our experimental setting, the risk of ASFV transmission via consumption of unprocessed insect larvae, used as feed, is low.

Evidence for emerging parasites and pathogens influencing outbreaks of stress-related diseases like chalkbrood.

In agriculture, honey bees play a critical role as commercial pollinators of crop monocultures which depend on insect pollination. Hence, the demise of honey bee colonies in Europe, USA, and Asia caused much concern and initiated many studies and research programmes aiming at elucidating the factors negatively affecting honey bee health and survival. Most of these studies look at individual factors related to colony losses. In contrast, we here present our data on the interaction of pathogens and parasites in honey bee colonies. We performed a longitudinal cohort study over 6 years by closely monitoring 220 honey bee colonies kept in 22 apiaries (ten randomly selected colonies per apiary). Observed winter colony losses varied between 4.8% and 22.4%; lost colonies were replaced to ensure a constant number of monitored colonies over the study period. Data on mite infestation levels, infection with viruses, Nosema apis and Nosema ceranae, and recorded outbreaks of chalkbrood were continuously collected. We now provide statistical evidence (i) that Varroa destructor infestation in summer is related to DWV infections in autumn, (ii) that V. destructor infestation in autumn is related to N. apis infection in the following spring, and most importantly (iii) that chalkbrood outbreaks in summer are related to N. ceranae infection in the preceding spring and to V. destructor infestation in the same season. These highly significant links between emerging parasites/pathogens and established pathogens need further experimental proof but they already illustrate the complexity of the host-pathogen-interactions in honey bee colonies.

Resistance and Vulnerability of Honeybee (Apis mellifera) Gut Bacteria to Commonly Used Pesticides.

Agricultural and apicultural practices expose honeybees to a range of pesticides that have the potential to negatively affect their physiology, neurobiology, and behavior. Accumulating evidence suggests that these effects extend to the honeybee gut microbiome, which serves important functions for honeybee health. Here we test the potential effects of the pesticides thiacloprid, acetamiprid, and oxalic acid on the gut microbiota of honeybees, first in direct in vitro inhibition assays and secondly in an in vivo caged bee experiment to test if exposure leads to gut microbiota community changes. We found that thiacloprid did not inhibit the honeybee core gut bacteria in vitro, nor did it affect overall community composition or richness in vivo. Acetamiprid did also not inhibit bacterial growth in vitro, but it did affect community structure within bees. The eight bacterial genera tested showed variable levels of susceptibility to oxalic acid in vitro. In vivo, treatment with this pesticide reduced amplicon sequence variant (ASV) richness and affected gut microbiome composition, with most marked impact on the common crop bacteria Lactobacillus kunkeei and the genus Bombella. We conducted network analyses which captured known associations between bacterial members and illustrated the sensitivity of the microbiome to environmental stressors. Our findings point to risks of honeybee exposure to oxalic acid, which has been deemed safe for use in treatment against Varroa mites in honeybee colonies, and we advocate for more extensive assessment of the long-term effects that it may have on honeybee health.

Nutritional Composition of Apis mellifera Drones from Korea and Denmark as a Potential Sustainable Alternative Food Source: Comparison Between Developmental Stages.

We compared nutrient compositions of honey bee (Apis mellifera) drones of different developmental stages from two different populations-the Italian honey bee reared in Korea and Buckfast bees from Denmark. Analyses included amino acid, fatty acid, and mineral content as well as evaluations of antioxidant properties and haemolysis activities. The compositions of total amino acids, and thus protein content of the insects, increased with development. A similar trend was observed for minerals presumably due to the consumption of food in the adult stage. In contrast, total fatty acid amounts decreased with development. Altogether, seventeen amino acids, including all the essential ones, except tryptophan, were determined. Saturated fatty acids dominated over monounsaturated fatty acids in the pupae, but the reverse held true for the adults. Drones were found to be rich in minerals and the particularly high iron as well as K/Na ratio was indicative of the nutritional value of these insects. Among the three developmental stages, adult Buckfast drones exhibited the highest antioxidant activity. Bearing in mind the overall high nutritional value, i.e., high amino acids, minerals and less fatty acids, late pupae and adult drones can be useful for human consumption while the larvae or early pupal stage can be recommended as feed. However, owing to their relatively high haemolysis activity, we advocate processing prior to the consumption of these insects.

Social immunity behaviour among ants infected by specialist and generalist fungi.

Social insects are distinguished by their lifestyle of living in groups with division of labour, cooperative brood care, and reproduction limited to a few colony members. Social insects often build large colonies with remarkable densities of highly related individuals and this can lead to an increased pathogen pressure. Our review focuses on interactions of ants with two important taxonomic groups of fungi infecting ants: Hypocreales (Ascomycota) and Entomophthorales (Entomophthoromycotina), and their different infection strategies, including host manipulation for optimal spore dispersal in the specialised ant pathogens. In social insects such as ants, resistance to pathogens is present at the colony level, with social immunity in addition to the individual resistance. We describe how ants use both organizational and behavioural defence strategies to combat fungal pathogens, with emphasis on highly specialised fungi from the genera Ophiocordyceps and Pandora.

Statistical modelling of conidial discharge of entomophthoralean fungi using a newly discovered Pandora species.

Entomophthoralean fungi are insect pathogenic fungi and are characterized by their active discharge of infective conidia that infect insects. Our aim was to study the effects of temperature on the discharge and to characterize the variation in the associated temporal pattern of a newly discovered Pandora species with focus on peak location and shape of the discharge. Mycelia were incubated at various temperatures in darkness, and conidial discharge was measured over time. We used a novel modification of a statistical model (pavpop), that simultaneously estimates phase and amplitude effects, into a setting of generalized linear models. This model is used to test hypotheses of peak location and discharge of conidia. The statistical analysis showed that high temperature leads to an early and fast decreasing peak, whereas there were no significant differences in total number of discharged conidia. Using the proposed model we also quantified the biological variation in the timing of the peak location at a fixed temperature.

Factors driving susceptibility and resistance in aphids that share specialist fungal pathogens.

Pandora neoaphidis and Entomophthora planchoniana are widespread and important specialist fungal pathogens of aphids in cereals (Sitobion avenae and Rhopalosiphum padi). The two aphid species share these pathogens and we compare factors influencing susceptibility and resistance. Among factors that may influence susceptibility and resistance are aphid behavior, conspecific versus heterospecific host, aphid morph, life cycle, and presence of protective endosymbionts. It seems that the conspecific host is more susceptible (less resistant) than the heterospecific host, and alates are more susceptible than apterae. We conceptualize the findings in a diagram showing possible transmission in field situations and we pinpoint where there are knowledge gaps.