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.

An enteric ultrastructural surface atlas of the model insect Manducasexta.

The tobacco hornworm is a laboratory model that is particularly suitable for analyzing gut inflammation, but a physiological reference standard is currently unavailable. Here, we present a surface atlas of the healthy hornworm gut generated by scanning electron microscopy and nano-computed tomography. This comprehensive overview of the gut surface reveals morphological differences between the anterior, middle, and posterior midgut, allowing the screening of aberrant gut phenotypes while accommodating normal physiological variations. We estimated a total resorptive midgut surface of 0.42 m2 for L5d6 larvae, revealing its remarkable size. Our data will support allometric scaling and dose conversion from Manduca sexta to mammals in preclinical research, embracing the 3R principles. We also observed non-uniform gut colonization by enterococci, characterized by dense biofilms in the pyloric cone and downstream of the pylorus associated with pore and spine structures in the hindgut intima, indicating a putative immunosurveillance function in the lepidopteran hindgut.

The insect microbiome is a vast source of bioactive small molecules.

Covering: September 1964 to June 2023Bacteria and fungi living in symbiosis with insects have been studied over the last sixty years and found to be important sources of bioactive natural products. Not only classic producers of secondary metabolites such as Streptomyces and other members of the phylum Actinobacteria but also numerous bacteria from the phyla Proteobacteria and Firmicutes and an impressive array of fungi (usually pathogenic) serve as the source of a structurally diverse number of small molecules with important biological activities including antimicrobial, cytotoxic, antiparasitic and specific enzyme inhibitors. The insect niche is often the exclusive provider of microbes producing unique types of biologically active compounds such as gerumycins, pederin, dinactin, and formicamycins. However, numerous insects still have not been described taxonomically, and in most cases, the study of their microbiota is completely unexplored. In this review, we present a comprehensive survey of 553 natural products produced by microorganisms isolated from insects by collating and classifying all the data according to the type of compound (rather than the insect or microbial source). The analysis of the correlations among the metadata related to insects, microbial partners, and their produced compounds provides valuable insights into the intricate dynamics between insects and their symbionts as well as the impact of their metabolites on these relationships. Herein, we focus on the chemical structure, biosynthesis, and biological activities of the most relevant compounds.

The Crayfish Plague Pathogen Aphanomyces astaci in Ireland.

Crayfish plague is a devastating disease of European freshwater crayfish and is caused by the oomycete Aphanomyces astaci (Ap. astaci), believed to have been introduced to Europe around 1860. All European species of freshwater crayfish are susceptible to the disease, including the white-clawed crayfish Austropotamobius pallipes. Ap. astaci is primarily spread by North American crayfish species and can also disperse rapidly through contaminated wet gear moved between water bodies. This spread, coupled with competition from non-indigenous crayfish, has drastically reduced and fragmented native crayfish populations across Europe. Remarkably, the island of Ireland remained free from the crayfish plague pathogen for over 100 years, providing a refuge for A. pallipes. However, this changed in 1987 when a mass mortality event was linked to the pathogen, marking its introduction to the region. Fortunately, crayfish plague was not detected again in Ireland until 2015 when a molecular analysis linked a mass mortality event in the Erne catchment to Ap. astaci. Since then, the pathogen has appeared across the island. Between 2015 and 2023, Ap. astaci was detected in 18 water catchments, revealing multiple genotypes. Intriguingly, the pathogen in Ireland is present without its natural host species. The uneven distribution of various genetic lineages strongly suggests the human-mediated transport of zoospores via contaminated water equipment as a primary cause of spread. This review details the timeline of these events, Ap. astaci's introduction into Ireland, and its rapid spread. As well, this review references the genotypes that have been determined, and discusses the issue of non-indigenous crayfish species in Ireland and management efforts.

Design of Polymer Carriers for Optimized Pheromone Release in Sustainable Insect Control Strategies.

Semiochemicals such as pheromones play a major role in communication between insects, influencing their spatial orientation, aggregation, defense, and mating. The rational chemical design of precision pheromone-releasing materials are increased the efficiency of pheromone-based plant protection agents. Decades of research is begun to unravel the complex communication structures regulated by semiochemicals, from the neuronal perception of specific chemical substances to the behavioral responses in hundreds of species, including many devastating pest insects. This article summarizes the most effective uses of semiochemicals in agriculture, the behavioral responses of selected target species, and controlled-release strategies based on formulations such as novel fibrous polymer carriers. This study helps scientists, decision-makers, farmers, and the public understand the importance of appropriate mating disruption techniques that reduce the need for broad-spectrum insecticides and limit their impact on non-target and beneficial insects.

The resuscitation-promoting factor (Rpf) from Micrococcus luteus and its putative reaction product 1,6-anhydro-MurNAc increase culturability of environmental bacteria.

Dormant bacterial cells do not divide and are not immediately culturable, but they persist in a state of low metabolic activity, a physiological state having clinical relevance, for instance in latent tuberculosis. Resuscitation-promoting factors (Rpfs) are proteins that act as signalling molecules mediating growth and replication. In this study we aimed to test the effect of Rpfs from Micrococcus luteus on the number and diversity of cultured bacteria using insect and soil samples, and to examine if the increase in culturability could be reproduced with the putative reaction product of Rpf, 1,6-anhydro-N-acetylmuramic acid (1,6-anhydro-MurNAc). The rpf gene from Micrococcus luteus was amplified and cloned into a pET21b expression vector and the protein was expressed in Escherichia coli BL21(DE3) cells and purified by affinity chromatography using a hexa-histidine tag. 1,6-Anhydro-MurNAc was prepared using reported chemical synthesis methods. Recombinant Rpf protein or 1,6-anhydro-MurNAc were added to R2A cultivation media, and their effect on the culturability of bacteria from eight environmental samples including four cockroach guts and four soils was examined. Colony-forming units, 16S rRNA gene copies and Illumina amplicon sequencing of the 16S rRNA gene were measured for all eight samples subjected to three different treatments: Rpf, 1,6-anhydro-MurNAc or blank control. Both Rpf and 1,6-anhydro-MurNAc increased the number of colony-forming units and of 16S rRNA gene copies across the samples although the protein was more effective. The Rpf and 1,6-anhydro-MurNAc promoted the cultivation of a diverse set of bacteria and in particular certain clades of the phyla Actinomycetota and Bacillota . This study opens the path for improved cultivation strategies aiming to isolate and study yet undescribed living bacterial organisms.

Prevalent bee venom genes evolved before the aculeate stinger and eusociality.

BACKGROUND: Venoms, which have evolved numerous times in animals, are ideal models of convergent trait evolution. However, detailed genomic studies of toxin-encoding genes exist for only a few animal groups. The hyper-diverse hymenopteran insects are the most speciose venomous clade, but investigation of the origin of their venom genes has been largely neglected. RESULTS: Utilizing a combination of genomic and proteo-transcriptomic data, we investigated the origin of 11 toxin genes in 29 published and 3 new hymenopteran genomes and compiled an up-to-date list of prevalent bee venom proteins. Observed patterns indicate that bee venom genes predominantly originate through single gene co-option with gene duplication contributing to subsequent diversification. CONCLUSIONS: Most Hymenoptera venom genes are shared by all members of the clade and only melittin and the new venom protein family anthophilin1 appear unique to the bee lineage. Most venom proteins thus predate the mega-radiation of hymenopterans and the evolution of the aculeate stinger.

Venomics of the milos viper (Macrovipera schweizeri) unveils patterns of venom composition and exochemistry across blunt-nosed viper venoms.

Introduction: Snakebite is a neglected tropical disease and a globally important driver of death and morbidity. Vipers of the genus Macrovipera (Viperidae: Viperinae) are among the snakes of higher medical importance in the Old World. Despite the medical relevance of Macrovipera venoms, the knowledge regarding them is heterogeneously distributed with virtually all works conducted so far focusing on subspecies of Macrovipera lebetinus, while other species within the genus are largely overlooked. Here we present the first proteomic evaluation of the venom from the Greek endemic Milos viper (Macrovipera schweizeri). In line with clinical symptoms typically elicited by Macrovipera envenomations, Milos viper venom primarily comprises coagulotoxic and cytotoxic protein families, such as metalloproteinases (svMP) and serine proteases (svSP). Methods: We conducted comparative bioactivity assays on venoms from M. schweizeri and the M. lebetinus subspecies M. lebetinus cernovi, M. lebetinus obtusa, and M. lebetinus turanica, and showed that they all exhibit similarities in levels of cytotoxicity proteolytic activity, and inhibition of prokaryotic growth. Lastly, we compared Macrovipera venom profiles by 1D-SDS-PAGE and RP-HPLC, as well as our proteomic data with previously published Macrovipera venom proteomes. Results and discussion: The analyzes performed to reveal that a general venom profile seems to be conserved across blunt-nosed vipers, and that, M. schweizeri envenomations, similarly to those caused by other blunt-nosed vipers, are able to cause significant tissue damage. The present work represents an important starting point for the development of comparative studies across the full taxonomic range of the genus Macrovipera and can potentially help optimize the treatment of envenomations caused by M. schweizeri.

Extracts of Talaromyces purpureogenus Strains from Apis mellifera Bee Bread Inhibit the Growth of Paenibacillus spp. In Vitro.

Honey bees coexist with fungi that colonize hive surfaces and pollen. Some of these fungi are opportunistic pathogens, but many are beneficial species that produce antimicrobial compounds for pollen conservation and the regulation of pathogen populations. In this study, we tested the in vitro antimicrobial activity of Talaromyces purpureogenus strains isolated from bee bread against Paenibacillus alvei (associated with European foulbrood disease) and three Aspergillus species that cause stonebrood disease. We found that methanol extracts of T. purpureogenus strains B18 and B195 inhibited the growth of P. alvei at a concentration of 0.39 mg/mL. Bioactivity-guided dereplication revealed that the activity of the crude extracts correlated with the presence of diketopiperazines, a siderophore, and three unknown compounds. We propose that non-pathogenic fungi such as Talaromyces spp. and their metabolites in bee bread could be an important requirement to prevent disease. Agricultural practices involving the use of fungicides can disrupt the fungal community and thus negatively affect the health of bee colonies.

A quantitative micro-tomographic gut atlas of the lepidopteran model insect Manduca sexta.

The tobacco hornworm is used extensively as a model system for ecotoxicology, immunology and gut physiology. Here, we established a micro-computed tomography approach based on the oral application of the clinical contrast agent iodixanol, allowing for a high-resolution quantitative analysis of the Manduca sexta gut. This technique permitted the identification of previously unknown and understudied structures, such as the crop or gastric ceca, and revealed the underlying complexity of the hindgut folding pattern, which is involved in fecal pellet formation. The acquired data enabled the volume rendering of all gut parts, the reliable calculation of their volumes, and the virtual endoscopy of the entire alimentary tract. It can provide information for accurate orientation in histology uses, enable quantitative anatomical phenotyping in three dimensions, and allow the calculation of locally effective midgut concentrations of applied chemicals. This atlas will provide critical insights into the evolution of the alimentary tract in lepidopterans.

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.

Full Profiling of GE81112A, an Underexplored Tetrapeptide Antibiotic with Activity against Gram-Negative Pathogens.

After the first total synthesis combined with structure revision, we performed thorough in vitro and in vivo profiling of the underexplored tetrapeptide GE81112A. From the determination of the biological activity spectrum and physicochemical and early absorption-distribution-metabolism-excretion-toxicity (eADMET) properties, as well as in vivo data regarding tolerability and pharmacokinetics (PK) in mice and efficacy in an Escherichia coli-induced septicemia model, we were able to identify the critical and limiting parameters of the original hit compound. Thus, the generated data will serve as the basis for further compound optimization programs and developability assessments to identify candidates for preclinical/clinical development derived from GE81112A as the lead structure. IMPORTANCE The spread of antimicrobial resistance (AMR) is becoming a more and more important global threat to human health. With regard to current medical needs, penetration into the site of infection represents the major challenge in the treatment of infections caused by Gram-positive bacteria. Considering infections associated with Gram-negative bacteria, resistance is a major issue. Obviously, novel scaffolds for the design of new antibacterials in this arena are urgently needed to overcome this crisis. Such a novel potential lead structure is represented by the GE81112 compounds, which inhibit protein synthesis by interacting with the small 30S ribosomal subunit using a binding site distinct from that of other known ribosome-targeting antibiotics. Therefore, the tetrapeptide antibiotic GE81112A was chosen for further exploration as a potential lead for the development of antibiotics with a new mode of action against Gram-negative bacteria.

Functional Profiling of the A-Family of Venom Peptides from the Wolf Spider Lycosa shansia.

The venoms of spiders from the RTA (retro-lateral tibia apophysis) clade contain diverse short linear peptides (SLPs) that offer a rich source of therapeutic candidates. Many of these peptides have insecticidal, antimicrobial and/or cytolytic activities, but their biological functions are unclear. Here, we explore the bioactivity of all known members of the A-family of SLPs previously identified in the venom of the Chinese wolf spider (Lycosa shansia). Our broad approach included an in silico analysis of physicochemical properties and bioactivity profiling for cytotoxic, antiviral, insecticidal and antibacterial activities. We found that most members of the A-family can form α-helices and resemble the antibacterial peptides found in frog poison. The peptides we tested showed no cytotoxic, antiviral or insecticidal activities but were able to reduce the growth of bacteria, including clinically relevant strains of Staphylococcus epidermidis and Listeria monocytogenes. The absence of insecticidal activity may suggest that these peptides have no role in prey capture, but their antibacterial activity may help to defend the venom gland against infection.

Venom biotechnology: casting light on nature's deadliest weapons using synthetic biology.

Venoms are complex chemical arsenals that have evolved independently many times in the animal kingdom. Venoms have attracted the interest of researchers because they are an important innovation that has contributed greatly to the evolutionary success of many animals, and their medical relevance offers significant potential for drug discovery. During the last decade, venom research has been revolutionized by the application of systems biology, giving rise to a novel field known as venomics. More recently, biotechnology has also made an increasing impact in this field. Its methods provide the means to disentangle and study venom systems across all levels of biological organization and, given their tremendous impact on the life sciences, these pivotal tools greatly facilitate the coherent understanding of venom system organization, development, biochemistry, and therapeutic activity. Even so, we lack a comprehensive overview of major advances achieved by applying biotechnology to venom systems. This review therefore considers the methods, insights, and potential future developments of biotechnological applications in the field of venom research. We follow the levels of biological organization and structure, starting with the methods used to study the genomic blueprint and genetic machinery of venoms, followed gene products and their functional phenotypes. We argue that biotechnology can answer some of the most urgent questions in venom research, particularly when multiple approaches are combined together, and with other venomics technologies.

An Assassin's Secret: Multifunctional Cytotoxic Compounds in the Predation Venom of the Assassin Bug Psytalla horrida (Reduviidae, Hemiptera).

Predatory assassin bugs produce venomous saliva that enables them to overwhelm, kill, and pre-digest large prey animals. Venom from the posterior main gland (PMG) of the African assassin bug Psytalla horrida has strong cytotoxic effects, but the responsible compounds are yet unknown. Using cation-exchange chromatography, we fractionated PMG extracts from P. horrida and screened the fractions for toxicity. Two venom fractions strongly affected insect cell viability, bacterial growth, erythrocyte integrity, and intracellular calcium levels in Drosophila melanogaster olfactory sensory neurons. LC-MS/MS analysis revealed that both fractions contained gelsolin, redulysins, S1 family peptidases, and proteins from the uncharacterized venom protein family 2. Synthetic peptides representing the putative lytic domain of redulysins had strong antimicrobial activity against Escherichia coli and/or Bacillus subtilis but only weak toxicity towards insect or mammalian cells, indicating a primary role in preventing the intake of microbial pathogens. In contrast, a recombinant venom protein family 2 protein significantly reduced insect cell viability but exhibited no antibacterial or hemolytic activity, suggesting that it plays a role in prey overwhelming and killing. The results of our study show that P. horrida secretes multiple cytotoxic compounds targeting different organisms to facilitate predation and antimicrobial defense.

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.

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.

Vagococcus luciliae sp. nov., isolated from the common green bottle fly Lucilia sericata.

The genus Vagococcus belongs to the family Enterococcaceae (order Lactobacillales) and is closely related to the genus Enterococcus. Currently, 19 species of the genus have been validly named. In this study, we isolated strain G314FT from the common green bottle fly Lucilia sericata collected in Germany. Sequencing of its almost-complete 16S rRNA gene revealed that the isolate belongs to the genus Vagococcus, being closely related to Vagococcus bubulae SS1994T with high sequence identity (99.50 %), followed by Vagococcus martis D7T301T (98.86 %), Vagococcus vulneris SS1995T (98.71 %), Vagococcus teuberi DSM 21459T (98.64 %), Vagococcus silagei 2B-2T (98.64 %) and Vagococcus penaei CD276T (98.64 %). Genome sequencing of strain G314FT was performed by a combination of Illumina and Oxford Nanopore technology, yielding a circular genome with a size of 2 139 468 bp and an 11 kb plasmid. Average nucleotide identity and digital DNA-DNA hybridization values were calculated between G314FT and its closest-related taxa, and found to be <91 % and <40 %, respectively, thus strongly supporting that strain G314FT represents a novel species of the genus Vagococcus. Phylogenetic and core protein-based phylogenomic trees revealed that G314FT was closely related to a group of three species, V. bubulae SS1994T, V. martis D7T301T and V. teuberi DSM 21459T. Comparatively, the genome of G314FT is the smallest in the group of the four related species, and the biochemical pathway comparison using BlastKOALA revealed that G314FT has lost some amino acid biosynthetic proteins; however, it has gained enzymes for carbohydrate metabolism. Phenotypically, G314FT was consistent with other species of the genus Vagococcus including a negative catalase reaction and non-motility. Using the polyphasic approach, our data supports that the isolate represents a new species, for which we propose the name Vagococcus luciliae G314FT (=DSM 112651T= CCM 9164T).

Spatial metabolomics reveal divergent cardenolide processing in the monarch (Danaus plexippus) and the common crow butterfly (Euploea core).

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini) is still unknown. While monarchs tolerate cardenolides by a resistant Na+ /K+ -ATPase, it is unclear how closely related species such as the nonsequestering common crow butterfly (Euploea core, Lepidoptera: Danaini) cope with these toxins. Using novel atmospheric-pressure scanning microprobe matrix-assisted laser/desorption ionization mass spectrometry imaging, we compared the distribution of cardenolides in caterpillars of D. plexippus and E. core. Specifically, we tested at which physiological scale quantitative differences between both species are mediated and how cardenolides distribute across body tissues. Whereas D. plexippus sequestered most cardenolides from milkweed (Asclepias curassavica), no cardenolides were found in the tissues of E. core. Remarkably, quantitative differences already manifest in the gut lumen: while monarchs retain and accumulate cardenolides above plant concentrations, the toxins are degraded in the gut lumen of crows. We visualized cardenolide transport over the monarch midgut epithelium and identified integument cells as the final site of storage where defences might be perceived by predators. Our study provides molecular insight into cardenolide sequestration and highlights the great potential of mass spectrometry imaging for understanding the kinetics of multiple compounds including endogenous metabolites, plant toxins, or insecticides in insects.