Azole resistance in Aspergillus flavus and associated fitness cost.

BACKGROUND: The resistance of Aspergillus flavus to the azole antifungal drugs is an emerging problem. Mutations in the molecular targets of the azole antifungals - CYP 51 A, B and C - are possible mechanisms of resistance, but data to confirm this hypothesis are scarce. In addition, the behaviour of resistant strains in vitro and in vivo is not yet understood. OBJECTIVES: This study had 3 objectives. The first was to compare the sequences of CYP51 A, B and C in resistant and susceptible strains of A. flavus. The second was to look for the existence of a fitness cost associated with resistance. The third was to evaluate the activity of voriconazole and posaconazole on resistant strains in the Galleria mellonella model. METHODS: The CYP51 A, B and C sequences of seven resistant strains with those of four susceptible strains are compared. Fitness costs were assessed by growing the strains in RPMI medium and testing their virulence in G. mellonella larvae. In addition, G. mellonella larvae infected with strains of A. flavus were treated with voriconazole and posaconazole. RESULTS: In the CYP51A sequences, we found the A91T, C708T and A1296T nucleotide substitutions only in the resistant strains. The resistant strains showed a fitness cost with reduced in vitro growth and reduced virulence in G. mellonella. In vivo resistance to posaconazole is confirmed in a strain with the highest MIC for this antifungal agent. CONCLUSIONS: These results allow to conclude that some substitutions in CYP51 genes, in particular CYP51A, contribute to resistance to azole drugs in A. flavus. The study of the relationship between drug dosage and treatment duration with resistance and the reduction of fitness costs in resistant strains is a major perspective of this study. This work could help to establish recommendations for the treatment of infections with resistant strains of A. flavus.

Suppressing the virulence factors of Candida auris with baicalein through multifaceted mechanisms.

Candida auris, a rapidly spreading multi-drug-resistant fungus, is causing lethal infections under certain conditions globally. Baicalin (BE), an active ingredient extracted from the dried root of Scutellaria baicalensis Georgi, exhibits antifungal activity. However, studies have shown the distinctive advantages of Traditional Chinese medicine in combating fungal infections, while the effect of BE, an active ingredient extracted from the dried roots of Scutellaria baicalensis Georgi, on C. auris, remains unknown. Therefore, this study aims to evaluate the potential of BE as an antifungal agent against the emerging multidrug-resistant C. auris. Various assays and models, including microbroth dilution, time growth curve analysis, spot assays, adhesion tests, flocculation test, cell surface hydrophobicity assay, hydrolase activity assays, XTT assay, violet crystal assay, scanning electron microscope (SEM), confocal laser scanning microscope (CLSM), flow cytometry, Live/dead fluorescent staining, reactive oxygen species (ROS), cell wall assay, aggregation assay, porcine skin model, Galleria mellonella larvae (G. mellonella larvae) infection model, and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were utilized to investigate how baicalein suppresses C. auris through possible multifaceted mechanisms. The findings indicate that BE strongly inhibited C. auris growth, adhesion, and biofilm formation. It also effectively reduced drug resistance and aggregation by disrupting the cell membrane and cell wall while reducing colonization and invasion of the host. Transcriptome analysis showed significant modulation in gene expression related to different virulence factors post-BE treatment. In conclusion, BE exhibits significant effectiveness against C. auris, suggesting its potential as a viable treatment option due to its multifaceted suppression mechanisms.

Using the Zebrafish Larval Model of Infection to Investigate Antibiotic Efficacy and Combination Treatments Against Staphylococcus aureus.

There is an increasing need for new treatment regimens to combat antibiotic-resistant strains of bacteria. Staphylococcus aureus is a clinically important, opportunist pathogen that has developed resistance to a range of antibiotics. The zebrafish larval model of systemic disease has been increasingly utilized to elucidate S. aureus virulence mechanisms and host-pathogen interactions. Here, we outline how this model can be used to investigate the effects of different antibiotics alone and in combination against S. aureus.

Effect of Microsporidia MB infection on the development and fitness of Anopheles arabiensis under different diet regimes.

BACKGROUND: Microsporidia MB (MB) is a naturally occurring symbiont of Anopheles and has recently been identified as having a potential to inhibit the transmission of Plasmodium in mosquitoes. MB intensity is high in mosquito gonads, with no fitness consequences for the mosquito, and is linked to horizontal (sexual) and vertical (transovarial) transmission from one mosquito to another. Maximising MB intensity and transmission is important for maintaining heavily infected mosquito colonies for experiments and ultimately for mosquito releases. We have investigated how diet affects the MB-Anopheles arabiensis symbiosis phenotypes, such as larval development and mortality, adult size and survival, as well as MB intensity in both larvae and adults. METHODS: F1 larvae of G0 females confirmed to be An. arabiensis and infected with MB were either combined (group lines [GLs]) or reared separately (isofemale lines [IMLs]) depending on the specific experiment. Four diet regimes (all mg/larva/day) were tested on F1 GLs: Tetramin 0.07, Tetramin 0.3, Gocat 0.3 and Cerelac 0.3. GLs reared on Tetramin 0.3 mg/larva/day were then fed either a 1% or 6% glucose diet to determine adult survival. Larvae of IMLs were fed Tetramin 0.07 mg and Tetramin 0.3 mg for larval experiments. The mosquitoes in the adult experiments with IMLs were reared on 1% or 6% glucose. RESULTS: Amongst the four larval diet regimes tested on An. arabiensis development in the presence of MB, the fastest larval development highest adult emergence, largest body size of mosquitoes, highest prevalence and highest density of MB occurred in those fed Tetramin 0.3 mg/larva/day. Although adult MB-positive mosquitoes fed on 6% glucose survived longer than MB-negative mosquitoes, there was no such effect for those fed on the 1% glucose diet. Development time, wing length and adult survival were not significantly different between MB-infected and uninfected An. arabiensis fed on the Tetramin 0.07 mg/larva/day diet, demonstrating that the MB-conferred fitness advantage was diet-dependent. CONCLUSIONS: Microsporidia MB does not adversely impact the development and fitness of An. arabiensis, even under limited dietary conditions. The diet regime of Tetramin 0.3 mg/larva/day + 6% glucose for adults is the superior diet for the mass rearing of MB-infected An. arabiensis mosquitoes. These results are important for rearing MB-infected An. arabiensis in the laboratory for experiments and the mass rearing required for field releases.

Microbiome Dynamics and Functional Composition in Coelopa frigida (Diptera, Coelopidae): Insights into Trophic Specialization of Kelp Flies.

Coelopidae (Diptera), known as kelp flies, exhibit an ecological association with beached kelp and other rotting seaweeds. This unique trophic specialization necessitates significant adaptations to overcome the limitations of an algal diet. We aimed to investigate whether the flies' microbiome could be one of these adaptive mechanisms. Our analysis focused on assessing composition and diversity of adult and larval microbiota of the kelp fly Coelopa frigida. Feeding habits of the larvae of this species have been subject of numerous studies, with debates whether they directly consume kelp or primarily feed on associated bacteria. By using a 16S rRNA metabarcoding approach, we found that the larval microbiota displayed considerably less diversity than adults, heavily dominated by only four operational taxonomic units (OTUs). Phylogenetic placement recovered the most dominant OTU of the larval microbiome, which is the source of more than half of all metabarcoding sequence reads, as an undescribed genus of Orbaceae (Gammaproteobacteria). Interestingly, this OTU is barely found among the 15 most abundant taxa of the adult microbiome, where it is responsible for less than 2% of the metabarcoding sequence reads. The other three OTUs dominating the larval microbiome have been assigned as Psychrobacter (Gammaproteobacteria), Wohlfahrtiimonas (Gammaproteobacteria), and Cetobacterium (Fusobacteriota). Moreover, we also uncovered a distinct shift in the functional composition between the larval and adult stages, where our taxonomic profiling suggests a significant decrease in functional diversity in larval samples. Our study offers insights into the microbiome dynamics and functional composition of Coelopa frigida.

Microbial influence on the larval survival of Japanese eel Anguilla japonica: Antibiotic-mediated alterations and biomarker isolation.

In rearing systems for the Japanese eel Anguilla japonica, although it is assumed that microorganisms influence larval survival and mortality, particularly during the early stages of growth, the effects of bacterial communities on larval survival have yet to be sufficiently determined. In this study, we compared the bacterial communities associated with larval survival at three stages of eel growth. To artificially alter bacterial communities and assess larval survival, eel larvae were treated with 11 types of antibiotic, and larval survival and bacterial characteristics were compared between the antibiotic-treated and antibiotic-free control groups. Throughout the three growth stages, eels treated with four antibiotics (polymyxin B, tetracycline, novobiocin, and erythromycin) had survival rates higher than those in the control groups. The bacterial communities of surviving larvae in the control and antibiotic groups and dead larvae in the control groups were subsequently analyzed using 16S rRNA gene amplicon sequencing. PERMANOVA analysis indicated that these three larval groups were characterized by significantly different bacterial communities. We identified 14 biomarker amplicon sequence variants (ASVs) of bacterial genera such as Oceanobacter, Alcanivorax, Marinobacter, Roseibium, and Sneathiella that were enriched in surviving larvae in the antibiotic treatment groups. In contrast, all four biomarker ASVs enriched in dead larvae of the control groups were from bacteria in the genus Vibrio. Moreover, 52 bacterial strains corresponding to nine biomarkers were isolated using a culture method. To the best of our knowledge, this is the first study to evaluate the bacterial communities associated with the survival and mortality of larvae in during the early stages of Japanese eel growth and to isolate biomarker bacterial strains. These findings will provide valuable insights for enhancing larval survival in the eel larval rearing systems from a microbiological perspective.

Ad libitum feeding of silkworm larvae powder-containing diets specifically influences metabolism-related and short-chain fatty acid-producing gut bacteria in mice.

Silkworm (Bombyx mori) larvae are expected to be useful as an ingredient in entomophagy. They are full of nutrients, including indigestible proteins; however, there have been few studies on the effects of the consumption of the entire body of silkworms on the intestinal microflora. We prepared a customized diet containing silkworm larval powder (SLP), and investigated the effects of ad libitum feeding of the SLP diet on the intestinal microbiota and the amount of short-chain fatty acids (SCFAs) in mice. We found that the diversity of the cecal and fecal microbiota increased in the mice fed the SLP diet (SLP group), and that the composition of their intestinal microbiota differed from that of the control mice. Furthermore, a genus-level microbiota analysis showed that in the SLP group, the proportions of Alistipes, Lachnospiraceae A2, and RF39, which are associated with the prevention of obesity, were significantly increased, while the proportions of Helicobacter and Anaerotruncus, which are associated with obesity, were significantly decreased. Additionally, the level of butyrate was increased in the SLP group, and Clostridia UCG 014 and Lachnospiraceae FCS020 were found to be associated with the level of butyrate, one of the major SCFAs. These findings indicated that silkworm powder may be useful as an insect food that might also improve obesity.

Temperatures above 37°C increase virulence of a convergent Klebsiella pneumoniae sequence type 307 strain.

Background: Convergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307. Methods: Hypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics. Results: Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways. Conclusion: This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.

Gut microbiota diversity in a dung beetle (Catharsius molossus) across geographical variations and brood ball-mediated microbial transmission.

The dung beetle primarily feeds on the feces of herbivorous animals and play a crucial role in ecological processes like material cycles and soil improvement. This study aims to explore the diversity and composition of the gut microbiota of Catharsius molossus (a renowned dung beetle originating from China and introduced to multiple countries for its ecological value) and exploring whether these gut microbes are transmitted vertically across generations. Using 16S rRNA and ITS rRNA gene sequencing techniques, we described the diversity and composition of gut microbes in C. molossus from different localities and different developmental stages (Egg, young larvae and old larvae). We discovered that the diversity of gut microbiota of dung beetles varied obviously among different geographical localities and different developmental stages, and we also discussed the potential influencing factors. Interestingly, the microbial community structure within the brood balls is more similar to male dung beetle than to that of females, which is consistent with the observation that the brood ball is constructed by the male dung beetle, with the female laying egg in it at the final step. This unique breeding method facilitates offspring in inheriting microbial communities from both the mother and the father. Initially, the larvae's gut microbiota closely mirrors that of the parental gift in these brood balls. As larvae grow, significant changes occur in their gut microbiota, including an increase in symbiotic bacteria like Lactococcus and Enterococcus. Analysis of the gut bacteria of adult dung beetles across various localities and different developmental stages identified nine core genera in adults, contributing to 67.80% of the total microbial abundance, and 11 core genera in beetles at different developmental stages, accounting for 49.13% of the total. Notably, seven genera were common between these two core groups. Our results suggest that Parental gifts can play a role in the vertical transmission of microbes, and the abundance of probiotics increases with larval development, supporting the hypothesis that "larval feeding behavior occurs in two stages: larvae first feed on parental gifts to acquire necessary microbes, then enrich symbiotic microbiota through consuming their own feces."

Evolutionary history of tyrosine-supplementing endosymbionts in pollen-feeding beetles.

Many insects feeding on nutritionally challenging diets like plant sap, leaves, or wood engage in ancient associations with bacterial symbionts that supplement limiting nutrients or produce digestive or detoxifying enzymes. However, the distribution, function, and evolutionary dynamics of microbial symbionts in insects exploiting other plant tissues or relying on a predacious diet remain poorly understood. Here, we investigated the evolutionary history and function of the intracellular gamma-proteobacterial symbiont "Candidatus Dasytiphilus stammeri" in soft-winged flower beetles (Coleoptera, Melyridae, Dasytinae) that transition from saprophagy or carnivory to palynivory (pollen-feeding) between larval and adult stage. Reconstructing the distribution of the symbiont within the Dasytinae phylogeny unraveled not only a long-term coevolution, originating from a single acquisition event with subsequent host-symbiont codiversification, but also several independent symbiont losses. The analysis of 20 different symbiont genomes revealed that their genomes are severely eroded. However, the universally retained shikimate pathway indicates that the core metabolic contribution to their hosts is the provisioning of tyrosine for cuticle sclerotization and melanization. Despite the high degree of similarity in gene content and order across symbiont strains, the capacity to synthesize additional essential amino acids and vitamins and to recycle urea is retained in some but not all symbionts, suggesting ecological differences among host lineages. This report of tyrosine-provisioning symbionts in insects with saprophagous or carnivorous larvae and pollen-feeding adults expands our understanding of tyrosine supplementation as an important symbiont-provided benefit across a broad range of insects with diverse feeding ecologies.

A rodent and tick bait for controlling white-footed mice (Peromyscus leucopus) and blacklegged ticks (Ixodes scapularis), the respective pathogen host and vector of the Lyme disease spirochetes.

A promising alternative approach to conventional vector and rodent control practices is the use of a bait containing a rodenticide and acaricide in controlling vectors and pathogen reservoirs concurrently. In the United States, Lyme disease continues to be the most prevalent vector-borne disease with approximately 500,000 Lyme disease cases estimated each year. Previous research has demonstrated the usefulness of a low dose fipronil bait in controlling Ixodes scapularis larvae feeding on white-footed mice. However, considering white-footed mice can be an unwanted species because of their association with tick-borne disease and hantaviruses, a combination rodent and tick bait (RTB) might provide a useful alternative to encourage additional community participation in integrated tick management (ITM) efforts. The purpose of this research was to evaluate the use of RTB (0.025 % warfarin, 0.005 % fipronil) in controlling white-footed mice and I. scapularis larvae. Studies were designed in part based on Environmental Protection Agency (EPA) guidelines. A laboratory choice test was conducted to evaluate the use of RTB in controlling white-footed mice over 15-day exposure when they were exposed to an alternative diet. Mice were observed every day for mortality and signs of warfarin toxicity. A simulated field test was conducted to evaluate the use of RTB, presented in the presence of an alternative diet, in controlling I. scapularis parasitizing white-footed mice over 4-day exposure. Mice were fitted with capsules and manually infested with I. scapularis larvae. The inside of each capsule was observed to evaluate tick attachment. Replete larvae detaching from each mouse were collected. Blood was collected from all treatment group mice via cardiac puncture to determine the fipronil sulfone concentration in plasma for each animal. Results indicated that RTB would be adequately consumed in the presence of an alternative diet under laboratory and simulated field conditions. Treatment with RTB resulted in 100 % mortality of white-footed mice during 15-day exposure and prevented 100 % larvae from feeding to repletion during 4-day exposure. All mice succumbing to RTB showed signs of warfarin toxicity. All mice parasitized with ticks that were exposed to RTB had fipronil sulfone detectable in plasma, with even the lowest concentration detected (8.1 parts per billion) controlling 100 % parasitizing I. scapularis larvae. The results suggest that RTB could be a useful means of rodent and tick control for use in ITM programs.

Limited impact of bacterial virulence on early mortality risk factors in Acinetobacter baumannii bacteremia observed in a Galleria mellonella model.

Acinetobacter baumannii (AB) has emerged as a major pathogen in vulnerable and severely ill patients. It remains unclear whether early mortality (EM) due to AB bacteremia is because of worse clinical characteristics of the infected patients or the virulence of the pathogen. In this study, we aimed to investigate the effect of AB virulence on EM due to bacteremia. This retrospective study included 138 patients with AB bacteremia (age: ≥ 18 years) who were admitted to a tertiary care teaching hospital in South Korea between 2015 and 2019. EM was defined as death occurring within 7 days of bacteremia onset. The AB clinical isolates obtained from the patients' blood cultures were injected into 15 Galleria mellonella larvae each, which were incubated for 5 days. Clinical isolates were classified into high- and low-virulence groups based on the number of dead larvae. Patients' clinical data were combined and subjected to multivariate Cox regression analyses to identify the risk factors for EM. In total, 48/138 (34.8%) patients died within 7 days of bacteremia onset. The Pitt bacteremia score was the only risk factor associated with EM. In conclusion, AB virulence had no independent effect on EM in patients with AB bacteremia.

Diversity of soil fungi and entomopathogenic fungi in subtropical mountain forest in southwest China.

Till now, the diversity of entomopathogenic fungi in subtropical mountain forest was less studied. Here, the vertical distribution of forest soil fungi, entomopathogenic fungi, and their environmental influencing factors in a subtropical mountain in western China were investigated. Soil samples were collected from four elevations in a subtropical forest in Shaanxi. The results indicated a greater richness of soil fungi at middle elevations and soil fungi were more even at low elevation. Soil pH, available iron, available potassium, total potassium, and available zinc were the most important influencing factors affecting this vertical distribution of fungi. Interestingly, the Isaria genus was predominant while Metarhizium and Beauveria showed decreasing abundance. The presence of Isaria showed a significant positive correlation with both total phosphorus and available iron, while, available zinc was negatively correlated. Metarhizium was influenced by elevation, pH, available phosphorus, and available copper and Beauveria was influenced by soil organic carbon, total nitrogen, total potassium, available potassium, and available zinc. Overall, as environmental factors affecting soil fungi, elevation, and plant species diversity were less important than soil physical and chemical properties. The virulence of isolated entomopathogenic fungi were tested against larvae of Tenebrio molitor, with mortality ranging from 31.11% to 100%. The above findings provide valuable data to deepen our understanding of the diversity of entomopathogenic fungi in subtropical mountain forests.

Protein Engineering of Vip3A in a Selected Bacillus thuringiensis Host for Consistent High Protein Production.

This study aimed to obtain reliable high Vip3A production from Bacillus thuringiensis (Bt) by modifying Vip3A to acquire higher thermostability in a suitable host. Bt117 is a great host for Vip3A production due to protein production consistency, low protease activity in culture media, and large amounts of mostly full-length protein, but it produces Vip3A with lower thermostability (Vip3Aa35). The C-terminal region of Bt117 Vip3A was replaced with that of a Vip3A with higher thermostability (Vip3Aa64 from Bt294) to generate the recombinant Bt117-Vip3Aa64-C. Like the parental strain Bt117, this strain expressed mostly full-length protein and exhibited low protease activity and similar protein expression profiles in culture media but retained greater larvicidal activity upon 37 °C storage like Bt294 Vip3Aa64. Importantly, every culture batch of Bt117-Vip3Aa64-C yielded over 200 mg/l Vip3A, which is a notable improvement over the original Vip3Aa64-producing strain Bt294 where 45% of culture batches failed to produce Vip3A at the same level. Successfully, we combined the superior qualities of two Bt strains, Bt294, which produces thermostable Vip3A but at low and inconsistent levels, and Bt117, which produces Vip3A with low thermostability but at consistently high levels. Protein engineering of Vip3A in Bt117 ultimately yielded an improved strain producing a thermostable Vip3A with reliably high protein production.

Comparative study of the gut microbial community structure of Spodoptera frugiperda and Spodoptera literal (Lepidoptera).

Background: Spodoptera frugiperda, the fall armyworm is a destructive invasive pest, and S. litura the tobacco cutworm, is a native species closely related to S. frugiperda. The gut microbiota plays a vital role in insect growth, development, metabolism and immune system. Research on the competition between invasive species and closely related native species has focused on differences in the adaptability of insects to the environment. Little is known about gut symbiotic microbe composition and its role in influencing competitive differences between these two insects. Methods: We used a culture-independent approach targeting the 16S rRNA gene of gut bacteria of 5th instar larvae of S. frugiperda and S. litura. Larvae were reared continuously on maize leaves for five generations. We analyzed the composition, abundance, diversity, and metabolic function of gut microbiomes of S. frugiperda and S. litura larvae. Results: Firmicutes, Proteobacteria, and Bacteroidetes were the dominant bacterial phyla in both species. Enterococcus, ZOR0006, Escherichia, Bacteroides, and Lactobacillus were the genera with the highest abundance in S. frugiperda. Enterococcus, Erysipelatoclostridium, ZOR0006, Enterobacter, and Bacteroides had the highest abundance in S. litura. According to α-diversity analysis, the gut bacterial diversity of S. frugiperda was significantly higher than that of S. litura. KEGG analysis showed 15 significant differences in metabolic pathways between S. frugiperda and S. litura gut bacteria, including transcription, cell growth and death, excretory system and circulatory system pathways. Conclusion: In the same habitat, the larvae of S. frugiperda and S. litura showed significant differences in gut bacterial diversity and community composition. Regarding the composition and function of gut bacteria, the invasive species S. frugiperda may have a competitive advantage over S. litura. This study provides a foundation for developing control strategies for S. frugiperda and S. litura.

The fungal protease BbAorsin contributes to growth, conidiation, germination, virulence, and antiphytopathogenic activities in Beauveria bassiana (Hypocreales: Cordycipitaceae).

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is one of the most destructive agricultural pests. The entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) is a biopesticide widely used for biocontrol of various pests. Secreted fungal proteases are critical for insect cuticle destruction and successful infection. We have previously shown that the serine protease BbAorsin in B. bassiana has entomopathogenic and antiphytopathogenic activities. However, the contribution of BbAorsin to fungal growth, conidiation, germination, virulence and antiphytopathogenic activities remains unclear. In this study, the deletion (ΔBbAorsin), complementation (Comp), and overexpression (BbAorsinOE) strains of B. bassiana were generated for comparative studies. The results showed that ΔBbAorsin exhibited slower growth, reduced conidiation, lower germination rate, and longer germination time compared to WT and Comp. In contrast, BbAorsinOE showed higher growth rate, increased conidiation, higher germination rate and shorter germination time. Injection of BbAorsinOE showed the highest virulence against S. frugiperda larvae, while injection of ΔBbAorsin showed the lowest virulence. Feeding BbAorsinOE resulted in lower pupation and adult eclosion rates and malformed adults. 16S rRNA sequencing revealed no changes in the gut microbiota after feeding either WT or BbAorsinOE. However, BbAorsinOE caused a disrupted midgut, leakage of gut microbiota into the hemolymph, and upregulation of apoptosis and immunity-related genes. BbAorsin can disrupt the cell wall of the phytopathogen Fusarium graminearum and alleviate symptoms in wheat seedlings and cherry tomatoes infected with F. graminearum. These results highlight the importance of BbAorsin for B. bassiana and its potential as a multifunctional biopesticide.

Bacterial c-di-GMP triggers metamorphosis of mussel larvae through a STING receptor.

Bacteria induced metamorphosis observed in nearly all marine invertebrates. However, the mechanism of bacteria regulating the larvae-juvenile metamorphosis remains unknown. Here, we test the hypothesis that c-di-GMP, a ubiquitous bacterial second-messenger molecule, directly triggers the mollusc Mytilus coruscus larval metamorphosis via the stimulator of interferon genes (STING) receptor. We determined that the deletion of c-di-GMP synthesis genes resulted in reduced c-di-GMP levels and biofilm-inducing activity on larval metamorphosis, accompanied by alterations in extracellular polymeric substances. Additionally, c-di-GMP extracted from tested varying marine bacteria all exhibited inducing activity on larval metamorphosis. Simultaneously, through pharmacological and molecular experiments, we demonstrated that M. coruscus STING (McSTING) participates in larval metamorphosis by binding with c-di-GMP. Our findings reveal that new role of bacterial c-di-GMP that triggers mussel larval metamorphosis transition, and extend knowledge in the interaction of bacteria and host development in marine ecosystems.

NagZ modulates the virulence of E. cloacae by acting through the gene of unknown function, ECL_03795.

ß-N-acetylglucosaminidase (NagZ), a cytosolic glucosaminidase, plays a pivotal role in peptidoglycan recycling. Previous research demonstrated that NagZ knockout significantly eradicated AmpC-dependent ß-lactam resistance in Enterobacter cloacae. However, NagZ's role in the virulence of E. cloacae remains unclear. Our study, incorporating data on mouse and Galleria mellonella larval mortality rates, inflammation markers, and histopathological examinations, revealed a substantial reduction in the virulence of E. cloacae following NagZ knockout. Transcriptome sequencing uncovered differential gene expression between NagZ knockout and wild-type strains, particularly in nucleotide metabolism pathways. Further investigation demonstrated that NagZ deletion led to a significant increase in cyclic diguanosine monophosphate (c-di-GMP) levels. Additionally, transcriptome sequencing and RT-qPCR confirmed significant differences in the expression of ECL_03795, a gene with an unknown function but speculated to be involved in c-di-GMP metabolism due to its EAL domain known for phosphodiesterase activity. Interestingly, in ECL_03795 knockout strains, a notable reduction in the virulence was observed, and virulence was rescued upon complementation with ECL_03795. Consequently, our study suggests that NagZ's function on virulence is partially mediated through the ECL_03795→c-di-GMP pathway, providing insight into the development of novel therapies and strongly supporting the interest in creating highly efficient NagZ inhibitors.

Chitinase Producing Gut-Associated Bacteria Affected the Survivability of the Insect Spodoptera frugiperda.

BACKGROUND: Fall armyworm (Spodoptera frugiperda) is a highly destructive maize pest that significantly threatens agricultural productivity. Existing control methods, such as chemical insecticides and entomopathogens, lack effectiveness, necessitating alternative approaches. METHODS: Gut-associated bacteria were isolated from the gut samples of fall armyworm and screened based on their chitinase and protease-producing ability before characterization through 16S rRNA gene sequence analysis. The efficient chitinase-producing Bacillus licheniformis FGE4 and Enterobacter cloacae FGE18 were chosen to test the biocontrol efficacy. As their respective cell suspensions and extracted crude chitinase enzyme, these two isolates were applied topically on the larvae, supplemented with their feed, and analyzed for their quantitative food use efficiency and survivability. RESULTS: Twenty-one high chitinase and protease-producing bacterial isolates were chosen. Five genera were identified by 16S rRNA gene sequencing: Enterobacter, Enterococcus, Bacillus, Pantoea, and Kocuria. In the biocontrol efficacy test, the consumption index and relative growth rate were lowered in larvae treated with Enterobacter cloacae FGE18 by topical application and feed supplementation. Similarly, topical treatment of Bacillus licheniformis FGE4 to larvae decreased consumption index, relative growth rate, conversion efficiency of ingested food, and digested food values. CONCLUSION: The presence of gut bacteria with high chitinase activity negatively affects insect health. Utilizing gut-derived bacterial isolates with specific insecticidal traits offers a promising avenue to control fall armyworms. This research suggests a potential strategy for future pest management.

Microplastics in the diet of Hermetia illucens: Implications for development and midgut bacterial and fungal microbiota.

In a world with a population exceeding 8 billion people and continuing to grow, pollution from food and plastic waste is causing long-term issues in ecosystems. Potential solutions may be found by exploiting insect-based bioconversion. In this context, we investigated the impact of polyvinyl chloride microparticles (PVC-MPs) on the development of Hermetia illucens (black soldier fly; BSF) and its midgut bacterial and fungal microbiota. The impact of PVC-MPs was evaluated feeding BSF larvae with a PVC-MPs-supplemented diet. The larvae exposed to different PVC-MPs concentrations (2.5%, 5%, 10% and 20% w/w) developed into adults with no significant increase in pupal mortality. Faster development and smaller pupae were observed when 20% PVC-MPs was provided. The BSF larvae ingest PVC-MPs, resulting in a reduction in MPs size. Larvae exposed to PVC-MPs did not exhibit differences in gut morphology. Regarding the impact of PVC-MPs on the structure of both bacterial and fungal communities, the overall alpha- and beta-diversity did not exhibit significant changes. However, the presence of PVC-MPs significantly affected the relative abundances of Enterobacteriaceae and Paenibacillaceae among the bacteria and of Dipodascaceae and Plectospharellaceae among the fungi (including yeast and filamentous life forms), suggesting that PVC-MP contamination has a taxa-dependent impact. These results indicate that BSF larvae can tolerate PVC-MPs in their diet, supporting the potential use of these insects in organic waste management, even in the presence of high levels of PVC-MP contamination.