Bacterial c-di-GMP signaling gene affects mussel larval metamorphosis through outer membrane vesicles and lipopolysaccharides.

Biofilms serve as crucial cues for settlement and metamorphosis in marine invertebrates. Within bacterial systems, c-di-GMP functions as a pivotal signaling molecule regulating both biofilm formation and dispersion. However, the molecular mechanism of how c-di-GMP modulates biofilm-induced larval metamorphosis remains elusive. Our study reveals that the deletion of a c-di-GMP related gene in Pseudoalteromonas marina led to an increase in the level of bacterial c-di-GMP by knockout technique, and the mutant strain had an enhanced ability to produce more outer membrane vesicles (OMVs) and lipopolysaccharides (LPS). The mutant biofilms had higher induction activity for larval metamorphosis in mussels Mytilus coruscus, and OMVs play a major role in the induction activity. We further explored the function of LPS in OMVs. Extracted LPS induced high larval metamorphosis rate, and LPS content were subject to c-di-GMP and LPS-biosynthesis gene. Thus, we postulate that the impact of c-di-GMP on biofilm-induced metamorphosis is mediated through OMVs and LPS.

Parasitism on domestic cats by Amblyomma auricularium and serological evidence of exposure to Rickettsia amblyommatis.

The domestic cat is not considered a primary host for any specific tick species; however, it can be affected by some Ixodidae species, such as Rhipicephalus sanguineus sensu lato and Amblyomma spp. The study reports parasitism by Amblyomma auricularium and the detection of anti-Rickettsia spp. antibodies in domestic cats from a rural property in the Afrânio municipality, Pernambuco, Brazil. Amblyomma auricularium (24 nymphs, six females, and four males) and Amblyomma sp. (42 larvae) parasitized three cats, and 73 free-living ticks were captured in armadillo burrows: A. auricularium (36 nymphs, six females, five males) and Amblyomma sp. (26 larvae). Blood samples from cats were collected and the obtained plasma were subjected to indirect immunofluorescence assay (IFA) to detect antibodies against Rickettsia antigens. Thus, anti-Rickettsia spp. antibodies were determined (titers ranging from 128 to 512) and showed a predominant antibody response to Rickettsia amblyommatis or a very closely related genotype. This study reports the first infestation of nymphs and adults of A. auricularium on cats in a new area of occurrence in the semi-arid region of Northeastern Brazil and reports for the first time the presence of anti-Ricketsia antibodies in cats in the region, with R. amblyommatis as the probable infectious agent.

Sensitive bioluminescence imaging of cryptococcosis in Galleria mellonella improves antifungal screening under in vivo conditions.

Cryptococcus neoformans is an environmental yeast that primarily affects immunocompromised individuals, causing respiratory infections and life-threatening meningoencephalitis. Treatment is complicated by limited antifungal options, with concerns such as adverse effects, dose-limiting toxicity, blood-brain barrier permeability, and resistance development, emphasizing the critical need to optimize and expand current treatment options against invasive cryptococcosis. Galleria mellonella larvae have been introduced as an ethical intermediate for in vivo testing, bridging the gap between in vitro antifungal screening and mouse studies. However, current infection readouts in G. mellonella are indirect, insensitive, or invasive, which hampers the full potential of the model. To address the absence of a reliable non-invasive method for tracking infection, we longitudinally quantified the cryptococcal burden in G. mellonella using bioluminescence imaging (BLI). After infection with firefly luciferase-expressing C. neoformans, the resulting bioluminescence signal was quantitatively validated using colony-forming unit analysis. Longitudinal comparison of BLI to health and survival analysis revealed increased sensitivity of BLI in discriminating cryptococcal burden during early infection. Furthermore, BLI improved the detection of treatment efficacy using first-line antifungals, thereby benchmarking this model for antifungal testing. In conclusion, we introduced BLI as a real-time, quantitative readout of cryptococcal burden in G. mellonella over time, enabling more sensitive and reliable antifungal screening.

Coconut rhinoceros beetle digestive symbiosis with potential plant cell wall degrading microbes.

Coconut rhinoceros beetle (CRB, Oryctes rhinoceros) is an invasive palm pest whose larvae eat wood, yet lack the necessary digestive enzymes. This study confirmed endogenous CRB cellulase is inactive, suggesting microbial fermentation. The inner lining of the CRB hindgut has tree-like structures covered with a conspicuous biofilm. To identify possible symbionts, 16 S rRNA amplicon sequencing was used on individuals from across Taiwan. Several taxa of Clostridia, an anaerobic class including many cellulolytic bacteria, were highly abundant in most individuals from all locations. Whole metagenome sequencing further confirmed many lignocellulose degrading enzymes are derived from these taxa. Analyses of eggs, larvae, adults, and soil found these cellulolytic microbes are not transmitted vertically or transstadially. The core microbiomes of the larval CRB are likely acquired and enriched from the environment with each molt, and enable efficient digestion of wood.

Dynamics and regulatory role of circRNAs in Asian honey bee larvae following fungal infection.

Non-coding RNA (ncRNA) plays a vital part in the regulation of immune responses, growth, and development in plants and animals. Here, the identification, characteristic analysis, and molecular verification of circRNAs in Apis cerana cerana worker larval guts were conducted, followed by in-depth investigation of the expression pattern of larval circRNAs during Ascosphaera apis infection and exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 3178 circRNAs in the larval guts of A. c. cerana were identified, with a length distribution ranging from 15 to 96,007 nt. Additionally, 155, 95, and 86 DEcircRNAs were identified in the in the 4-, 5-, and 6-day-old larval guts following A. apis infection. These DEcircRNAs were predicted to target 29, 25, and 18 parental genes relevant to 12, 20, and 17 GO terms as well as 144, 114, and 61 KEGG pathways, including 5 cellular and 4 humoral immune pathways. Complex competing endogenous RNA (ceRNA) regulatory networks were detected as being formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The target DEmRNAs were engaged in 36, 47, and 47 GO terms as well as 331, 332, and 331 pathways, including 6 cellular and 6 humoral immune pathways. Further, 19 DEcircRNAs, 5 DEmiRNAs, and 3 mRNAs were included in the sub-networks relative to 3 antioxidant enzymes. Finally, back-splicing sites within 15 circRNAs and the difference in the 15 DEcircRNAs' expression between uninoculated and A. apis-inoculated larval guts were confirmed based on molecular methods. These findings not only enrich our understanding of bee host-fungal pathogen interactions but also lay a foundation for illuminating the mechanism underlying the DEcircRNA-mediated immune defense of A. c. cerana larvae against A. apis invasion. KEY POINTS: • The expression pattern of circRNAs was altered in the A. cerana worker larval guts following A. apis infection. • Back-splicing sites within 15 A. cerana circRNAs were verified using molecular approaches. DEcircRNAs potentially modulated immune responses and antioxidant enzymes in A. apis-challenged host guts.

Density and behavior of capybara (Hydrochoerus hydrochaeris) ticks (Acari: Ixodidae) Amblyomma sculptum and Amblyomma dubitatum with notes on Rickettsia bellii infection: Assessing human exposure risk.

In several urban and peri­urban areas of Brazil, populations of Amblyomma sculptum and Amblyomma dubitatum ticks are maintained by capybaras (Hydrochoerus hydrochaeris). In some of these areas, this host and these tick species are associated with Brazilian spotted fever (BSF), a lethal human disease caused by the bacterium Rickettsia rickettsii. In this work, we evaluated the risk of human exposure to these tick species using four collection techniques to discern host-seeking behavior. The study was carried out in 10 urban sites inhabited by capybaras in Uberlândia, a BSF-free municipality in southeastern Brazil. Ticks were collected in areas of 400 m2 at each site and at three seasons. Within the same municipality, the distance and speed of A. sculptum nymphs moving towards the CO2 traps were evaluated. In a sample of ticks Rickettsia DNA was investigated. During the study period, 52,953 ticks were collected. Among these, 83.4 % were A. sculptum (1,523 adults, 10,545 nymphs and 32,104 larvae) and 16.6 % were A. dubitatum (464 adults, 2,153 nymphs and 6,164 larvae). An average annual questing tick density of 4.4/m² was observed, with the highest density recorded at one site in autumn (31.8/m²) and the lowest in summer at another site (0.03/m²). The visual search yielded the highest proportion of A. sculptum larvae, constituting 47 % of the total and 63.6 % of all A. sculptum larvae. In contrast, CO2 traps collected a greater proportion of nymphs and adults of A. sculptum ticks. In the case of A. dubitatum, the CO2 trap was the most efficient technique with 57.7 % of captures of this species, especially of nymphs (94.5 % of captures) and adults (97.8 % of captures). Ticks' ambush height on vegetation (9 to 77 cm), observed by visual search 30 times, yielded a total of 20,771 ticks. Of these, 28 (93 %) were A. sculptum ticks, with only two (7 %) identified as A. dubitatum ticks. Among A. sculptum ticks, the nymph was the most attracted stage to humans and larva in the case of A. dubitatum. Amblyomma sculptum adults and nymphs were significantly more attracted to humans than those of A. dubitatum, but A. dubitatum larvae were significantly more attracted than the same stage of A. sculptum. The maximum distance and speed of horizontal displacement for A. sculptum nymphs were five meters and 2.0 m/h, respectively. The only species of Rickettsia detected in ticks, exclusively in A. dubitatum, was R. bellii. Importantly, it was observed that the higher the proportion of A. sculptum in the community of ticks, the lower the rate of infection of A. dubitatum by R. bellii. In conclusion, host-seeking behavior differed between the two tick species, as well as between stages of the same species. A greater restriction of A. dubitatum ticks to the soil was observed, while larvae and nymphs of A. sculptum dispersed higher in the vegetation. The behavior presented by A. sculptum provides greater opportunities for contact with the hosts, while A. dubitatum depends more on an active search for a host, the hunter behavior. Taken together, these observations show that a human being crossing an area infested with A. sculptum and A. dubitatum ticks will have almost exclusive contact with A. sculptum larvae and/or nymphs. Humans in a stationary position (sitting, lying or immobile) are exposed to both tick species, but they are more attractive to adults and mainly nymphs of A. sculptum compared to the corresponding stages of the tick A. dubitatum. The negative effect of A. sculptum on A. dubitatum infection by R. bellii deserves further studies.

Enhancement of an entomopathogenic fungal virulence against the seedcorn maggot, Delia platura, by suppressing immune responses with a bacterial culture broth of Photorhabdus temperata subsp. temperata.

In Korea, there are two maggot species in the Delia genus that commonly infest the roots and stems of the Welsh onion, thus causing serious economic damage on the crop at the seedling stage. In this study, the seedcorn maggot (Delia platura) was detected in onion fields in two different localities in Korea. After overwintering, maggot infestations occurred throughout the entire growing seasons from transplantation to harvest, but their specific patterns of occurrence varied in the two localities examined. Entomopathogenic fungi induced significant virulence against the maggot larvae, in which a strain of Beauveria bassiana was effective, though it exhibited limited mortality in its insecticidal activity. To enhance this insecticidal activity, a culture broth from an entomopathogenic bacterium, Photorhabdus temperata temperata (Ptt), was added to B. bassiana treatment. The addition of Ptt broth significantly increased the insecticidal activity of B. bassiana in a dose-dependent manner. To elucidate this enhancement in insecticidal activity, the immunosuppressive activity of Ptt broth was assessed by identifying the immune responses of the seedcorn maggots. The seedcorn maggots possessed at least three different hemocytes with plasmatocytes, crystal cells, and lamellocytes. These hemocytes exhibited nodule formation in response to the fungal infection. In addition to the cellular immunity, the maggots exhibited inducible expressions of antimicrobial peptide (AMP) genes such as cecropin and defensin. The addition of Ptt broth suppressed the nodule formation and the AMP expressions in response to the fungal infection. Altogether, this study demonstrated the innate immune responses in a non-model insect, D. platura, along with the application of immunosuppression to develop a highly efficient biological control by enhancing the virulence of B. bassiana.

METARHIZIUM ANISOPLIAE SENSU LATO NATIVE TO LIVESTOCK SOILS CAUSES HIGH MORTALITY ON RHIPICEPHALUS MICROPLUS LARVAE, ADULTS AND AFFECTS THEIR REPRODUCTION.

The acaricidal effect of 14 strains of Metarhizium anisopliae sensu lato isolated from soil of livestock farms in the Mexican tropics was evaluated against larvae and engorged females, and during the laying and hatching of eggs of Rhipicephalus microplus (Ixodida: Ixodidae). For each fungal strain, the larvae mortality percentage was evaluated through a larval immersion test, while the reproductive efficiency indices in engorged females were measured using adult immersion tests at a dose of 1 × 108 conidia/ml. All strains of M. anisopliae (s.l.) proved to be highly effective against R. microplus larvae (66-100%) and engorged females (100%). The strains also showed a good effect in inhibiting egg laying (16.45-56.38%) and a moderate effect in decreasing egg hatching (5.24-32.68%). Two strains demonstrated to be effective against all development phases of R. microplus in an integrated manner.

Dietary potential of the symbiotic fungus Penicillium herquei for the larvae of a nonsocial fungus-cultivating weevil Euops chinensis.

Many insect taxa cultivate fungi for food. Compared to well-known fungus cultivation in social insects, our knowledge on fungus cultivation in nonsocial insects is still limited. Here, we studied the nutritional potentials of the fungal cultivar, Penicillium herquei, for the larvae of its nonsocial insect farmer, Euops chinensis, a specialist on Japanese knotweed Reynoutria japonica. Overall, fungal hyphae and leaf rolls contained significantly higher carbon (C), stable isotopes of C (δ13C), and nitrogen (δ15N) but significantly lower C/N ratios compared to unrolled leaves, whereas insect bodies contained significantly higher N contents but lower C and C/N ratios compared to other types of samples. The MixSIAR model indicated that fungal hyphae contributed a larger proportion (0.626-0.797) to the diet of E. chinensis larvae than leaf materials. The levels of ergosterol, six essential amino acids, seven nonessential amino acids, and three B vitamins tested in fungal hyphae and/or leaf rolls were significantly higher than in unrolled leaves and/or larvae. The P. herquei genome contains the complete set of genes required for the biosynthesis of ergosterol, the essential amino acids valine and threonine, nine nonessential amino acids, and vitamins B2 and B3, whereas some genes associated with five essential and one nonessential amino acid were lost in the P. herquei genome. These suggest that P. herquei is capable of providing the E. chinensis larvae food with ergosterol, amino acids, and B vitamins. P. herquei appears to be able to synthesize or concentrate these nutrients considering that they were specifically concentrated in fungal hyphae. IMPORTANCE: The cultivation of fungi for food has occurred across divergent insect lineages such as social ants, termites, and ambrosia beetles, as well as some seldom-reported solitary insects. Although the fungal cultivars of these insects have been studied for decades, the dietary potential of fungal cultivars for their hosts (especially for those nonsocial insects) is largely unknown. Our research on the mutualistic system Euops chinensis-Penicillium herquei represents an example of the diverse nutritional potentials of the fungal cultivar P. herquei in the diet of the larvae of its solitary host, E. chinensis. These results demonstrate that P. herquei has the potential to synthesize or concentrate ergosterol, amino acids, and B vitamins and benefits the larvae of E. chinensis. Our findings would shed light on poorly understood fungal cultivation mutualisms in nonsocial insects and underscore the nutritional importance of fungal cultivars in fungal cultivation mutualisms.

Entomopathogenic pseudomonads can share an insect host with entomopathogenic nematodes and their mutualistic bacteria.

A promising strategy to overcome limitations in biological control of insect pests is the combined application of entomopathogenic pseudomonads (EPPs) and nematodes (EPNs) associated with mutualistic bacteria (NABs). Yet, little is known about interspecies interactions such as competition, coexistence, or even cooperation between these entomopathogens when they infect the same insect host. We investigated the dynamics of bacteria-bacteria interactions between the EPP Pseudomonas protegens CHA0 and the NAB Xenorhabdus bovienii SM5 isolated from the EPN Steinernema feltiae RS5. Bacterial populations were assessed over time in experimental systems of increasing complexity. In vitro, SM5 was outcompeted when CHA0 reached a certain cell density, resulting in the collapse of the SM5 population. In contrast, both bacteria were able to coexist upon haemolymph-injection into Galleria mellonella larvae, as found for three further EPP-NAB combinations. Finally, both bacteria were administered by natural infection routes i.e. orally for CHA0 and nematode-vectored for SM5 resulting in the addition of RS5 to the system. This did not alter bacterial coexistence nor did the presence of the EPP affect nematode reproductive success or progeny virulence. CHA0 benefited from RS5, probably by exploiting access routes formed by the nematodes penetrating the larval gut epithelium. Our results indicate that EPPs are able to share an insect host with EPNs and their mutualistic bacteria without major negative effects on the reproduction of any of the three entomopathogens or the fitness of the nematodes. This suggests that their combination is a promising strategy for biological insect pest control.

An obligate microsporidian parasite modulates defense against opportunistic bacterial infection in the yellow fever mosquito, Aedes aegypti.

The ability of Aedes aegypti mosquitoes to transmit vertebrate pathogens depends on multiple factors, including the mosquitoes' life history traits, immune response, and microbiota (i.e., the microbes associated with the mosquito throughout its life). The microsporidium Edhazardia aedis is an obligate intracellular parasite that specifically infects Ae. aegypti mosquitoes and severely affects mosquito survival and other life history traits critical for pathogen transmission. In this work, we investigated how E. aedis impacts bacterial infection with Serratia marcescens in Ae. aegypti mosquitoes. We measured development, survival, and bacterial load in both larval and adult stages of mosquitoes. In larvae, E. aedis exposure was either horizontal or vertical and S. marcescens was introduced orally. Regardless of the route of transmission, E. aedis exposure resulted in significantly higher S. marcescens loads in larvae. E. aedis exposure also significantly reduced larval survival but subsequent exposure to S. marcescens had no effect. In adult females, E. aedis exposure was only horizontal and S. marcescens was introduced orally or via intrathoracic injection. In both cases, E. aedis infection significantly increased S. marcescens bacterial loads in adult female mosquitoes. In addition, females infected with E. aedis and subsequently injected with S. marcescens suffered 100% mortality which corresponded with a rapid increase in bacterial load. These findings suggest that exposure to E. aedis can influence the establishment and/or replication of other microbes in the mosquito. This has implications for understanding the ecology of mosquito immune defense and potentially disease transmission by mosquito vector species. IMPORTANCE: The microsporidium Edhazardia aedis is a parasite of the yellow fever mosquito, Aedes aegypti. This mosquito transmits multiple viruses to humans in the United States and around the world, including dengue, yellow fever, and Zika viruses. Hundreds of millions of people worldwide will become infected with one of these viruses each year. E. aedis infection significantly reduces the lifespan of Ae. aegypti and is therefore a promising novel biocontrol agent. Here, we show that when the mosquito is infected with this parasite, it is also significantly more susceptible to infection by an opportunistic bacterial pathogen, Serratia marcescens. This novel discovery suggests the mosquito's ability to control infection by other microbes is impacted by the presence of the parasite.

Effects of antimicrobial peptides from dietary Hermetia illucens larvae on the growth, immunity, gene expression, intestinal microbiota and resistance to Aeromonas hydrophila of juvenile red claw crayfish (Cherax quadricarinatus).

Antimicrobial peptides (AMPs), which are widely present in animals and plants, have a broad distribution, strong broad-spectrum antibacterial activity, low likelihood of developing drug resistance, high thermal stability and antiviral properties. The present study investigated the effects of adding AMPs from Hermetia illucens larvae on the growth performance, muscle composition, antioxidant capacity, immune response, gene expression, antibacterial ability and intestinal microbiota of Cherax quadricarinatus (red claw crayfish). Five experimental diets were prepared by adding 50 (M1), 100 (M2), 150 (M3) and 200 (M4) mg/kg of crude AMP extract from H. illucens larvae to the basal diet feed, which was also used as the control (M0). After an eight-week feeding experiment, it was discovered that the addition of 100-150 mg/kg of H. illucens larvae AMPs to the feed significantly improved the weight gain rate and specific growth rate of C. quadricarinatus. Furthermore, the addition of H. illucens larvae AMPs to the feed had no significant effect on the moisture content, crude protein, crude fat and ash content of the C. quadricarinatus muscle. The addition of 100-150 mg/kg of H. illucens larvae AMPs in the feed also increased the antioxidant capacity, nonspecific immune enzyme activity and related gene expression levels in C. quadricarinatus, thereby enhancing their antioxidant capacity and immune function. The H. illucens larvae AMPs improved the structure and composition of the intestinal microbiota of C. quadricarinatus, increasing the microbial community diversity of the crayfish gut. Finally, the addition of 100-150 mg/kg of H. illucens larvae AMPs in the feed enhanced the resistance of C. quadricarinatus against Aeromonas hydrophila, improving the survival rate of the crayfish. Based on the aforementioned findings, it is recommended that H. illucens larvae AMPs be incorporated into the C. quadricarinatus feed at a concentration of 100-150 mg/kg.

Bacterial Strains Isolated from Stingless Bee Workers Inhibit the Growth of Apis mellifera Pathogens.

Apis mellifera bees are an important resource for the local economy of various regions in Argentina and the maintenance of natural ecosystems. In recent years, different alternatives have been investigated to avoid the reduction or loss of colonies caused by pathogens and parasites such as Ascosphaera apis, Aspergillus flavus, and Paenibacillus larvae. We focused on bacterial strains isolated from the intestine of native stingless bees, to elucidate their antagonistic effect on diseases of A. mellifera colonies. For this purpose, worker bees of the species Tetragonisca fiebrigi, Plebeia spp., and Scaptotrigona jujuyensis were captured from the entrance to tree hives and transported to the laboratory, where their intestines were extracted. Twenty bacterial colonies were isolated from the intestines, and those capable of inhibiting enterobacteria in vitro and producing organic acids, proteases, and chitinases were selected. Four genera, Levilactobacillus, Acetobacter, Lactiplantibacillus, and Pantoea, were selected and identified by the molecular marker that codes for the 16S rRNA gene. For inhibition assays, cell suspensions and cell-free suspensions were performed. All treatments showed significant antibacterial effects, in comparison with the controls, against P. larvae and antifungal effects against A. apis and A. flavus. However, the mechanisms by which these bacteria inhibit the growth of these pathogens were not studied.

Something in the water: aquatic microbial communities influence the larval amphibian gut microbiota, neurodevelopment and behaviour.

Microorganisms colonize the gastrointestinal tract of animals and establish symbiotic host-associated microbial communities that influence vertebrate physiology. More specifically, these gut microbial communities influence neurodevelopment through the microbiota-gut-brain (MGB) axis. We tested the hypothesis that larval amphibian neurodevelopment is affected by the aquatic microbial community present in their housing water. Newly hatched Northern Leopard Frog (Lithobates pipiens) tadpoles were raised in pond water that was unmanipulated (natural) or autoclaved. Tadpoles raised in autoclaved pond water had a gut microbiota with reduced bacterial diversity and altered community composition, had decreased behavioural responses to sensory stimuli, were larger in overall body mass, had relatively heavier brains and had altered brain shape when compared with tadpoles raised in natural pond water. Further, the diversity and composition of the gut microbiota were associated with tadpole behavioural responses and brain measurements. Our results suggest that aquatic microbial communities shape tadpole behaviour and brain development, providing strong support for the occurrence of the MGB axis in amphibians. Lastly, the dramatic role played by aquatic microbial communities on vertebrate neurodevelopment and behaviour should be considered in future wildlife conservation efforts.

Abietic Acid as a Novel Agent against Ocular Biofilms: An In Vitro and Preliminary In Vivo Investigation.

Biofilm-related ocular infections can lead to vision loss and are difficult to treat with antibiotics due to challenges with application and increasing microbial resistance. In turn, the design and testing of new synthetic drugs is a time- and cost-consuming process. Therefore, in this work, for the first time, we assessed the in vitro efficacy of the plant-based abietic acid molecule, both alone and when introduced to a polymeric cellulose carrier, against biofilms formed by Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in standard laboratory settings as well as in a self-designed setting using the topologically challenging surface of the artificial eye. These analyses were performed using the standard microdilution method, the biofilm-oriented antiseptic test (BOAT), a modified disk-diffusion method, and eyeball models. Additionally, we assessed the cytotoxicity of abietic acid against eukaryotic cell lines and its anti-staphylococcal efficacy in an in vivo model using Galleria mellonella larvae. We found that abietic acid was more effective against Staphylococcus than Pseudomonas (from two to four times, depending on the test applied) and that it was generally more effective against the tested bacteria (up to four times) than against the fungus C. albicans at concentrations non-cytotoxic to the eukaryotic cell lines and to G. mellonella (256 and 512 µg/mL, respectively). In the in vivo infection model, abietic acid effectively prevented the spread of staphylococcus throughout the larvae organisms, decreasing their lethality by up to 50%. These initial results obtained indicate promising features of abietic acid, which may potentially be applied to treat ocular infections caused by pathogenic biofilms, with higher efficiency manifested against bacterial than fungal biofilms.

From phyllosphere to insect cuticles: silkworms gather antifungal bacteria from mulberry leaves to battle fungal parasite attacks.

BACKGROUND: Bacterial transfers from plants to insect herbivore guts have been well investigated. However, bacterial exchanges between plant phyllospheres and insect cuticles remain unclear, as does their related biological function. RESULTS: Here, we report that the cuticular bacterial loads of silkworm larvae quickly increased after molting and feeding on the white mulberry (Morus alba) leaves. The isolation and examination of silkworm cuticular bacteria identified one bacterium Mammaliicoccus sciuri that could completely inhibit the spore germination of fungal entomopathogens Metarhizium robertsii and Beauveria bassiana. Interestingly, Ma. sciuri was evident originally from mulberry leaves, which could produce a secreted chitinolytic lysozyme (termed Msp1) to damage fungal cell walls. In consistency, the deletion of Msp1 substantially impaired bacterial antifungal activity. Pretreating silkworm larvae with Ma. sciuri cells followed by fungal topical infections revealed that this bacterium could help defend silkworms against fungal infections. Unsurprisingly, the protective efficacy of ΔMsp1 was considerably reduced when compared with that of wild-type bacterium. Administration of bacterium-treated diets had no negative effect on silkworm development; instead, bacterial supplementation could protect the artificial diet from Aspergillus contamination. CONCLUSIONS: The results of this study evidence that the cross-kingdom transfer of bacteria from plant phyllospheres to insect herbivore cuticles can help protect insects against fungal parasite attacks. Video Abstract.

Chytridiomycosis causes high amphibian mortality prior to the completion of metamorphosis.

Amphibian populations are undergoing extensive declines globally. The fungal disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is a primary contributor to these declines. The amphibian metamorphic stages (Gosner stages 42-46) are particularly vulnerable to a range of stressors, including Bd. Despite this, studies that explicitly examine host response to chytridiomycosis throughout the metamorphic stages are lacking. We aimed to determine how Bd exposure during the larval stages impacts metamorphic development and infection progression in the endangered Fleay's barred frog (Mixophyes fleayi). We exposed M. fleayi to Bd during pro-metamorphosis (Gosner stages 35-38) and monitored infection dynamics throughout metamorphosis. We took weekly morphological measurements (weight, total body length, snout-vent-length and Gosner stage) and quantified Bd load using qPCR. While we observed minimal impact of Bd infection on animal growth and development, Bd load varied throughout ontogeny, with an infection load plateau during the tadpole stages (Gosner stages 35-41) and temporary infection clearance at Gosner stage 42. Bd load increased exponentially between Gosner stages 42 and 45, with most exposed animals becoming moribund at Gosner stage 45, prior to the completion of metamorphosis. There was variability in infection outcome of exposed individuals, with a subgroup of animals (n = 5/29) apparently clearing their infection while the majority (n = 21/29) became moribund with high infection burdens. This study demonstrates the role that metamorphic restructuring plays in shaping Bd infection dynamics and raises the concern that substantial Bd-associated mortality could be overlooked in the field due to the often cryptic nature of these latter metamorphic stages. We recommend future studies that directly examine the host immune response to Bd infection throughout metamorphosis, incorporating histological and molecular methods to elucidate the mechanisms responsible for the observed trends.

Simultaneous PCR detection of Paenibacillus larvae targeting insertion sequence IS256 and Melissococcus plutonius targeting pMP1 plasmid from hive specimens.

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

Bbhox2 is a key regulator for conidiation and virulence in Beauveria bassiana.

Beauveria bassiana, a well-known filamentous biocontrol fungus, is the main pathogen of numerous field and forest pests. To explore the potential factors involved in the fungal pathogenicity, Bbhox2, an important and conserved functional transcription factor containing homeodomain was carried out by functional analysis. Homologous recombination was used to disrupt the Bbhox2 gene in B.bassiana. The conidia yield of the deletant fungal strain was significantly reduced. The conidial germination was faster, and stress tolerance to Congo red and high osmotic agents were decreased compared with that in the wildtype. Additionally, ΔBbhox2 showed a dramatic reduction in virulence no matter in topical inoculations or in intra-hemolymph injections against Galleria mellonella larvae, which is likely due to the failure of appressorium formation and the defect in producing hyphal body. These results indicate that the Bbhox2 gene markedly contributes to conidiation and pathogenicity in B. bassiana.

Bacteriophage-mediated decolonization of Klebsiella pneumoniae in a novel Galleria mellonella gut colonization model with Enterobacteriaceae.

Galleria mellonella larvae have emerged as an invertebrate model for investigating bacterial pathogenesis and potential therapies, addressing ethical concerns related to mammalian models. This model has the advantage of having a simple gut microbiome, which is suitable for gut colonization studies. Intestinal colonization by Enterobacteriaceae significantly contributes to the spread of antibiotic resistance. This study aimed to establish a novel Enterobacteriaceae gut colonization larval model and assess its suitability for evaluating distinct antimicrobial efficacies. Larvae were force-fed sequentially with bacterial doses of K. pneumoniae and E. coli at 0, 24, and 48 h, with survival monitoring at 24 h intervals. Bacterial counts were assessed after 48 h and 120 h of force-feeding. Successfully colonized larvae were subjected to one-time force feeding of a bacteriophage cocktail (107 PFU/larvae) or MIC-based meropenem and ciprofloxacin. The colonized bacterial load was quantified by CFU count. Three doses of 106 CFU/larvae resulted in stable gut colonization, independent of the K. pneumoniae or E. coli strain. Compared with the control, force-feeding of the bacteriophage reduced the colonization of the strain Kp 419614 by 5 log10 CFU/larvae, while antibiotic treatment led to a 3 log10 CFU/larval reduction. This novel G. mellonella model provides a valuable alternative for gut colonization studies, facilitating proof-of-concept investigations and potentially reducing or replacing follow-up experiments in vertebrate models.