Identification of entomopathogenic bacteria associated with the invasive pest Drosophila suzukii in infested areas of Germany.

The invasive insect pest Drosophila suzukii causes extensive damage to soft-skinned fruit crops as they ripen. Current control methods involve the application of chemical pesticides, but this approach is ineffective and environmentally hazardous. To investigate the potential of bacterial pathogens carried by D. suzukii as biocontrol agents, we characterized bacteria associated with D. suzukii larvae in two parts of Hesse, Germany, by collecting infested fruits and culturing individual bacteria from moribund specimens for taxonomic classification by 16S rDNA sequencing. Among the bacteria we detected, some had a detrimental effect on the host whereas others were neutral or beneficial. When the detrimental and beneficial bacteria were presented simultaneously, we observed complex tripartite interactions that modulated the insect's innate immune response. Our study provides insight into the complex relationships within the microbiome and pathobiome of D. suzukii and may lead to the isolation of bacteria that can be used as biological control agents.

Identification and characterization of natural viruses associated with the invasive insect pest Drosophila suzukii.

The invasive insect pest Drosophila suzukii infests ripening fruits and causes extensive damage to crops in the northern hemisphere. Novel, environmentally friendly strategies to control the spread of this species are urgently needed, and one promising approach is the deployment of entomopathogenic viruses. Here we report the identification and characterization of two natural viruses associated with D. suzukii: Drosophila A virus (DAV) and La Jolla virus (LJV). Our work provides new tools for the development of biological control agents that protect crops against D. suzukii without a harmful impact on biodiversity.

Analysis of virus susceptibility in the invasive insect pest Drosophila suzukii.

The invasive insect pest Drosophila suzukii infests ripening fruits and causes massive agricultural damage in North America and Europe (Cini et al., 2012). Environmentally sustainable strategies are urgently needed to control the spread of this species, and entomopathogenic viruses offer one potential solution for global crop protection. Here we report the status of intrinsic and extrinsic factors that influence the susceptibility of D. suzukii to three model insect viruses: Drosophila C virus, Cricket paralysis virus and Flock house virus. Our work provides the basis for further studies using D. suzukii as a host system to develop viruses as biological control agents.

Drosophila C virus systemic infection leads to intestinal obstruction.

UNLABELLED: Drosophila C virus (DCV) is a positive-sense RNA virus belonging to the Dicistroviridae family. This natural pathogen of the model organism Drosophila melanogaster is commonly used to investigate antiviral host defense in flies, which involves both RNA interference and inducible responses. Although lethality is used routinely as a readout for the efficiency of the antiviral immune response in these studies, virus-induced pathologies in flies still are poorly understood. Here, we characterize the pathogenesis associated with systemic DCV infection. Comparison of the transcriptome of flies infected with DCV or two other positive-sense RNA viruses, Flock House virus and Sindbis virus, reveals that DCV infection, unlike those of the other two viruses, represses the expression of a large number of genes. Several of these genes are expressed specifically in the midgut and also are repressed by starvation. We show that systemic DCV infection triggers a nutritional stress in Drosophila which results from intestinal obstruction with the accumulation of peritrophic matrix at the entry of the midgut and the accumulation of the food ingested in the crop, a blind muscular food storage organ. The related virus cricket paralysis virus (CrPV), which efficiently grows in Drosophila, does not trigger this pathology. We show that DCV, but not CrPV, infects the smooth muscles surrounding the crop, causing extensive cytopathology and strongly reducing the rate of contractions. We conclude that the pathogenesis associated with systemic DCV infection results from the tropism of the virus for an important organ within the foregut of dipteran insects, the crop. IMPORTANCE: DCV is one of the few identified natural viral pathogens affecting the model organism Drosophila melanogaster. As such, it is an important virus for the deciphering of host-virus interactions in insects. We characterize here the pathogenesis associated with DCV infection in flies and show that it results from the tropism of the virus for an essential but poorly characterized organ in the digestive tract, the crop. Our results may have relevance for other members of the Dicistroviridae, some of which are pathogenic to beneficial or pest insect species.

Radioactivity and GMO-Free Sterile Insect Technology for the Sustainable Control of the Invasive Pest Drosophila suzukii.

Drosophila suzukii (D. suzukii), commonly known as the spotted wing drosophila, is a highly invasive crop pest that is difficult to control using chemical insecticides. To address the urgent need for alternative and more sustainable control strategies, the sterile insect technique (SIT) is improved, which involves the release of sterilized male insects to mate with fertile conspecifics, thereby reducing the size of the pest population in the subsequent generation. The three critical aspects that influence the success of SIT programs in D. suzukii are addressed. First, an accurate and nondestructive method is established to determine the sex of individual insects based on the differential weight of male and female pupae. Second, conditions for X-ray sterilization are systematically tested and an optimal dose (90 kV/40 Gy) is identified that ensures the efficient production of sterile D. suzukii for release. Finally, the inherent thermosensitivity of D. suzukii males is exploited to develop a temperature-based sterilization technique, offering an alternative or additional SIT method for this pest. These advances will contribute to the development of a comprehensive and effective strategy for the management of D. suzukii populations, reducing their impact on agriculture and helping to safeguard crop yields.

Prevalence in Potato of 'Candidatus Arsenophonus Phytopathogenicus' and 'Candidatus Phytoplasma Solani' and Their Transmission via Adult Pentastiridius leporinus.

The planthopper Pentastiridius leporinus (Hempiptera: Cixiidae) is the main vector of two bacterial pathogens: the γ-proteobacterium 'Candidatus Arsenophonus phytopathogenicus' and the stolbur phytoplasma 'Candidatus Phytoplasma solani'. These pathogens cause the disease syndrome basses richesses (SBR) in sugar beet (Beta vulgaris), which reduces the yields and sugar content. In 2022, potato (Solanum tuberosum) fields were found to be colonized by P. leporinus, and the transmission of Arsenophonus was confirmed, resulting in symptoms like wilting, yellow leaves, and rubbery tubers. We monitored both pathogens in Southwest Germany in 2022 and 2023. This revealed their widespread presence in potato tubers, although there were differences in regional prevalence. The broad prevalence of Arsenophonus was maintained in 2023, whereas the prevalence of stolbur increased in most locations. We confirmed that P. leporinus adults can transmit both pathogens to potatoes, but neither pathogen reduced the germination rate of tubers, and no plants showed abnormal growth after germination. Arsenophonus was not detected in germinated shoots, but 5.4% contained stolbur, emphasizing the need for plant material testing to maintain phytosanitary conditions.

Potato (Solanum tuberosum) as a New Host for Pentastiridius leporinus (Hemiptera: Cixiidae) and Candidatus Arsenophonus Phytopathogenicus.

Pentastiridius leporinus is a planthopper (Hemiptera: Cixiidae) that vectors two phloem-restricted bacterial pathogens to sugar beet (Beta vulgaris (L.)): the γ-proteobacterium Candidatus Arsenophonus phytopathogenicus and the stolbur phytoplasma Candidatus Phytoplasma solani. These bacteria cause an economically important disease known as syndrome basses richesses (SBR), characterized by yellowing, deformed leaves and low beet yields. Having observed potato fields in Germany infested with cixiid planthoppers and showing signs of leaf yellowing, we used morphological criteria and COI and COII as molecular markers, to identify the planthoppers (adults and nymphs) primarily as P. leporinus. We analyzed planthoppers, potato tubers, and sugar beet roots and detected both pathogens in all sample types, confirming that P. leporinus adults and nymphs can transmit the bacteria. This is the first time that P. leporinus has been shown to transmit Arsenophonus to potato plants. We also found that two generations of P. leporinus were produced in the warm summer of 2022, which will probably increase the pest population size (and thus the prevalence of SBR) in 2023. We conclude that P. leporinus has expanded its host range to potato, and can now utilize both host plants during its developmental cycle, a finding that will facilitate the development of more efficient control strategies.

Crude Extracts of Talaromyces Strains (Ascomycota) Affect Honey Bee (Apis mellifera) Resistance to Chronic Bee Paralysis Virus.

Viruses contribute significantly to the global decline of honey bee populations. One way to limit the impact of such viruses is the introduction of natural antiviral compounds from fungi as a component of honey bee diets. Therefore, we examined the effect of crude organic extracts from seven strains of the fungal genus Talaromyces in honey bee diets under laboratory conditions. The strains were isolated from bee bread prepared by honey bees infected with chronic bee paralysis virus (CBPV). The antiviral effect of the extracts was also quantified in vitro using mammalian cells as a model system. We found that three extracts (from strains B13, B18 and B30) mitigated CBPV infections and increased the survival rate of bees, whereas other extracts had no effect (B11 and B49) or were independently toxic (B69 and B195). Extract B18 inhibited the replication of feline calicivirus and feline coronavirus (FCoV) in mammalian cells, whereas extracts B18 and B195 reduced the infectivity of FCoV by ~90% and 99%, respectively. Our results show that nonpathogenic fungi (and their products in food stores) offer an underexplored source of compounds that promote disease resistance in honey bees.

Fast drosophila enterocyte regrowth after infection involves a reverse metabolic flux driven by an amino acid transporter.

Upon exposure to a bacterial pore-forming toxin, enterocytes rapidly purge their apical cytoplasm into the gut lumen, resulting in a thin intestinal epithelium. The enterocytes regain their original shape and thickness within 16 h after the ingestion of the bacteria. Here, we show that the regrowth of Drosophila enterocytes entails an inversion of metabolic fluxes from the organism back toward the intestine. We identify a proton-assisted transporter, Arcus, that is required for the reverse absorption of amino acids and the timely recovery of the intestinal epithelium. Arcus is required for a peak of amino acids appearing in the hemolymph shortly after infection. The regrowth of enterocytes involves the insulin signaling pathway and Myc. The purge decreases Myc mRNA levels, which subsequently remain at low levels in the arcus mutant. Interestingly, the action of arcus and Myc in the intestinal epithelium is not cell-autonomous, suggesting amino acid fluxes within the intestinal epithelium.

Peptidomics as a Tool to Assess the Cleavage of Wine Haze Proteins by Peptidases from Drosophila suzukii Larvae.

Thermolabile grape berry proteins such as thaumatin-like proteins (TLPs) and chitinases (CHIs) promote haze formation in bottled wines if not properly fined. As a natural grapevine pest, the spotted-wing fly Drosophila suzukii is a promising source of peptidases that break down grape berry proteins because the larvae develop and feed inside mature berries. Therefore, we produced recombinant TLP and CHI as model thermolabile wine haze proteins and applied a peptidomics strategy to investigate whether D. suzukii larval peptidases were able to digest them under acidic conditions (pH 3.5), which are typically found in winemaking practices. The activity of the novel peptidases was confirmed by mass spectrometry, and cleavage sites within the wine haze proteins were visualized in 3D protein models. The combination of recombinant haze proteins and peptidomics provides a valuable screening tool to identify optimal peptidases suitable for clarification processes in the winemaking industry.

RNA interference to combat the Asian tiger mosquito in Europe: A pathway from design of an innovative vector control tool to its application.

The Asian tiger mosquito Aedes albopictus is currently spreading across Europe, facilitated by climate change and global transportation. It is a vector of arboviruses causing human diseases such as chikungunya, dengue hemorrhagic fever and Zika fever. For the majority of these diseases, no vaccines or therapeutics are available. Options for the control of Ae. albopictus are limited by European regulations introduced to protect biodiversity by restricting or phasing out the use of pesticides, genetically modified organisms (GMOs) or products of genome editing. Alternative solutions are thus urgently needed to avoid a future scenario in which Europe faces a choice between prioritizing human health or biodiversity when it comes to Aedes-vectored pathogens. To ensure regulatory compliance and public acceptance, these solutions should preferably not be based on chemicals or GMOs and must be cost-efficient and specific. The present review aims to synthesize available evidence on RNAi-based mosquito vector control and its potential for application in the European Union. The recent literature has identified some potential target sites in Ae. albopictus and formulations for delivery. However, we found little information concerning non-target effects on the environment or human health, on social aspects, regulatory frameworks, or on management perspectives. We propose optimal designs for RNAi-based vector control tools against Ae. albopictus (target product profiles), discuss their efficacy and reflect on potential risks to environmental health and the importance of societal aspects. The roadmap from design to application will provide readers with a comprehensive perspective on the application of emerging RNAi-based vector control tools for the suppression of Ae. albopictus populations with special focus on Europe.

Culex Y Virus: A Native Virus of Culex Species Characterized In Vivo.

Mosquitoes are vectors of various pathogens that cause diseases in humans and animals. To prevent the outbreak of mosquito-borne diseases, it is essential to control vector populations, as treatment or vaccination for mosquito-borne diseases are often unavailable. Insect-specific viruses (ISVs) have previously been described as being potentially helpful against arboviral disease outbreaks. In this study, we present the first in vivo characterization of the ISV Culex Y virus (CYV). CYV was first isolated from free-living Culex pipiens mosquitoes in 2010; then, it was found in several mosquito cell lines in a further study in 2018. For mammalian cells, we were able to confirm that CYV does not replicate as it was previously described. Additionally, we found that CYV does not replicate in honey bees or locusts. However, we detected replication in the Culex pipiens biotype molestus, Aedes albopictus, and Drosophila melanogaster, thus indicating dipteran specificity. We detected significantly higher mortality in Culex pipiens biotype molestus males and Drosophila melanogaster, but not in Aedes albopictus and female Culex pipiens biotype molestus. CYV could not be transmitted transovarially to offspring, but we detected venereal transmission as well as CYV in mosquitos' saliva, indicating that an oral route of infection would also be possible. CYV's dipteran specificity, transmission routes, and killing effect with respect to Culex males may be used as powerful tools with which to destabilize arbovirus vector populations in the future.

Pathogenicity of La Jolla Virus in Drosophila suzukii following Oral Administration.

Drosophila suzukii (Ds) is an invasive pest insect that causes severe and widespread damage to soft fruit crops. Chemical control based on topical insecticides is inefficient and harmful to consumers and the environment, prompting interest in the development of biological control measures such as insect viruses with narrow host specificity. We previously described a strain of La Jolla virus (LJV) found in moribund Ds specimens in Germany. We demonstrated a pathogenic effect following the intrathoracic injection of LJV into adult Ds flies. However, the development of an effective biocontrol product based on LJV would require the characterization of (1) virulence following oral delivery, particularly in larvae, and (2) stability under different pH and temperature conditions reflecting realistic exposure scenarios. Here we describe the pathogenicity of LJV following oral delivery to Ds adults and larvae. The oral infection of Ds adults with LJV reduced survival in a concentration-dependent manner, whereas the oral infection of Ds larvae caused the arrest of development during pupation. LJV remained stable and infectious following exposure to a broad pH range and different temperatures. We, therefore, demonstrated that LJV is promising as a candidate biological control agent against Ds.

Biology and Rearing of an Emerging Sugar Beet Pest: The Planthopper Pentastiridius leporinus.

The rapid spread of the bacterial yellowing disease Syndrome des Basses Richesses (SBR) has a major impact on sugar beet (Beta vulgaris) cultivation in Germany, resulting in significant yield losses. SBR-causing bacteria are transmitted by insects, mainly the Cixiid planthopper Pentastiridius leporinus. However, little is known about the biology of this emerging vector, including its life cycle, oviposition, developmental stages, diapauses, and feeding behavior. Continuous mass rearing is required for the comprehensive analysis of this insect. Here we describe the development of mass rearing techniques for P. leporinus, allowing us to investigate life cycle and ecological traits, such as host plant choice, in order to design agronomic measures that can interrupt the life cycle of nymphs in the soil. We also conducted field studies in recently-infected regions of Rhineland-Palatinate and south Hesse, Germany, to study insect mobility patterns and abundance at four locations during two consecutive years. The soil-depth monitoring of nymphs revealed the movement of the instars through different soil layers. Finally, we determined the prevalence of SBR-causing bacteria by designing TaqMan probes specific for two bona fide SBR pathogens: Candidatus Arsenophonus phytopathogenicus (Gammaproteobacteria) and Candidatus Phytoplasma solani (stolbur phytoplasma). Our data suggest that P. leporinus is spreading northward and eastward in Germany, additionally, the abundance of SBR-carrying planthoppers is increasing. Interestingly, P. leporinus does not appear to hibernate during winter, and is polyphagous as a nymph. Stolbur phytoplasma has a significant impact on SBR pathology in sugar beet.

Exposure to low doses of pesticides induces an immune response and the production of nitric oxide in honeybees.

Honeybees are essential pollinators of many agricultural crops and wild plants. However, the number of managed bee colonies has declined in some regions of the world over the last few decades, probably caused by a combination of factors including parasites, pathogens and pesticides. Exposure to these diverse biotic and abiotic stressors is likely to trigger immune responses and stress pathways that affect the health of individual honeybees and hence their contribution to colony survival. We therefore investigated the effects of an orally administered bacterial pathogen (Pseudomonas entomophila) and low-dose xenobiotic pesticides on honeybee survival and intestinal immune responses. We observed stressor-dependent effects on the mean lifespan, along with the induction of genes encoding the antimicrobial peptide abaecin and the detoxification factor cytochrome P450 monooxygenase CYP9E2. The pesticides also triggered the immediate induction of a nitric oxide synthase gene followed by the delayed upregulation of catalase, which was not observed in response to the pathogen. Honeybees therefore appear to produce nitric oxide as a specific defense response when exposed to xenobiotic stimuli. The immunity-related and stress-response genes we tested may provide useful stressor-dependent markers for ecotoxicological assessment in honeybee colonies.

Organization of the Structural Protein Region of La Jolla Virus Isolated from the Invasive Pest Insect Drosophila suzukii.

Drosophila suzukii (Ds) is an invasive pest insect that infests ripening fruit, causing severe economic losses. Control measures based on chemical pesticides are inefficient and undesirable, so biological alternatives have been considered, including native Ds viruses. We previously isolated a strain of La Jolla virus (LJV-Ds-OS20) from Ds in Germany as a candidate biopesticide. Here we characterized the new strain in detail, focusing on the processing of its capsid proteins. We tested LJV growth during Ds development to optimize virus production, and established a laboratory production system using adult flies. This system was suitable for the preparation of virions for detailed analysis. The LJV-Ds-OS20 isolate was cloned by limiting dilution and the complete nucleotide sequence was determined as a basis for protein analysis. The terminal segments of the virus genome were completed by RACE-PCR. LJV virions were also purified by CsCl gradient centrifugation and analyzed by SDS-PAGE and electron microscopy. The capsid proteins of purified LJV virions were resolved by two-dimensional SDS-PAGE for N-terminal sequencing and peptide mass fingerprinting. The N-terminal sequences of VP1 and VP2, together with MS data representing several capsid proteins, allowed us to develop a model for the organization of the LJV structural protein region. This may facilitate the development of new viral strains as biopesticides.

The Cellular Innate Immune Response of the Invasive Pest Insect Drosophila suzukii against Pseudomonas entomophila Involves the Release of Extracellular Traps.

Drosophila suzukii is a neobiotic invasive pest that causes extensive damage to fruit crops worldwide. The biological control of this species has been unsuccessful thus far, in part because of its robust cellular innate immune system, including the activity of professional phagocytes known as hemocytes and plasmatocytes. The in vitro cultivation of primary hemocytes isolated from D. suzukii third-instar larvae is a valuable tool for the investigation of hemocyte-derived effector mechanisms against pathogens such as wasp parasitoid larvae, bacteria, fungi and viruses. Here, we describe the morphological characteristics of D. suzukii hemocytes and evaluate early innate immune responses, including extracellular traps released against the entomopathogen Pseudomonas entomophila and lipopolysaccharides. We show for the first time that D. suzukii plasmatocytes cast extracellular traps to combat P. entomophila, along with other cell-mediated reactions, such as phagocytosis and the formation of filopodia.

The Gram-Positive Bacterium Leuconostoc pseudomesenteroides Shows Insecticidal Activity against Drosophilid and Aphid Pests.

Insect pests reduce global crop yields by up to 20%, but the most effective control measures are currently based on environmentally hazardous chemical pesticides. An alternative, ecologically beneficial pest-management strategy involves the use of microbial pathogens (or active compounds and extracts derived from them) that naturally target selected insect pests. A novel strain of the bacterium Leuconostoc pseudomesenteroides showed promising activity in our preliminary tests. Here, we investigated its effects in more detail, focusing on drosophilid and aphid pests by testing the survival of two species representing the family Drosophilidae (Drosophila suzukii and D. melanogaster) and one representing the family Aphididae (Acyrthosiphon pisum). We used oral and septic infection models to administer living bacteria or cell-free extracts to adult flies and aphid nymphs. We found that infection with living bacteria significantly reduced the survival of our insect models, whereas the administration of cell-free extracts had a significant effect only in aphids. These results confirm that L. pseudomesenteroides has potential as a new biocontrol agent for sustainable pest management.

Sub-Lethal Doses of Clothianidin Inhibit the Conditioning and Biosensory Abilities of the Western Honeybee Apis mellifera.

Insects play an important role in the stability of ecosystems by fulfilling key functions such as pollination and nutrient cycling, as well as acting as prey for amphibians, reptiles, birds and mammals. The global decline of insects is therefore a cause for concern, and the role of chemical pesticides must be examined carefully. The lethal effects of insecticides are well understood, but sub-lethal concentrations have not been studied in sufficient detail. We therefore used the western honeybee Apis mellifera as a model to test the effect of the neonicotinoid insecticide clothianidin on the movement, biosensory abilities and odor-dependent conditioning of insects, titrating from lethal to sub-lethal doses. Bees treated with sub-lethal doses showed no significant movement impairment compared to untreated control bees, but their ability to react to an aversive stimulus was inhibited. These results show that clothianidin is not only highly toxic to honeybees, but can, at lower doses, also disrupt the biosensory capabilities of survivors, probably reducing fitness at the individual level. In our study, sub-lethal doses of clothianidin altered the biosensory abilities of the honeybee; possible consequences at the colony level are discussed.