Biological activity of silver nanoparticles synthesized using viticultural waste.

This research paper presents a novel approach to the green synthesis of silver nanoparticles (AgNPs) using viticultural waste, allowing to obtain NP dispersions with distinct properties and morphologies (monodisperse and polydisperse AgNPs, referred to as mAgNPs and pAgNPs) and to compare their biological activities. Our synthesis method utilized the ethanolic extract of Vitis vinifera pruning residues, resulting in the production of mAgNPs and pAgNPs with average sizes of 12 ± 5 nm and 19 ± 14 nm, respectively. Both these AgNPs preparations demonstrated an exceptional stability in terms of size distribution, which was maintained for one year. Antimicrobial testing revealed that both types of AgNPs inhibited either the growth of planktonic cells or the metabolic activity of biofilm sessile cells in Gram-negative bacteria and yeasts. No comparable activity was found towards Gram-positives. Overall, pAgNPs exhibited a higher antimicrobial efficacy compared to their monodisperse counterparts, suggesting that their size and shape may provide a broader spectrum of interactions with target cells. Both AgNP preparations showed no cytotoxicity towards a human keratinocyte cell line. Furthermore, in vivo tests using a silkworm animal model indicated the biocompatibility of the phytosynthesized AgNPs, as they had no adverse effects on insect larvae viability. These findings emphasize the potential of targeted AgNPs synthesized from viticultural waste as environmentally friendly antimicrobial agents with minimal impact on higher organisms.

Selective breeding of cold-tolerant black soldier fly (Hermetia illucens) larvae: Gut microbial shifts and transcriptional patterns.

The larvae of black soldier fly (BSFL) convert organic waste into insect proteins used as feedstuff for livestock and aquaculture. BSFL production performance is considerably reduced during winter season. Herein, the intraspecific diversity of ten commercial BSF colonies collected in China was evaluated. The Bioforte colony was subjected to selective breeding at 12 °C and 16 °C to develop cold-tolerant BSF with improved production performance. After breeding for nine generations, the weight of larvae, survival rate, and the dry matter conversion rate significantly increased. Subsequently, intestinal microbiota in the cold-tolerant strain showed that bacteria belonging to Morganella, Dysgonomonas, Salmonella, Pseudochrobactrum, and Klebsiella genera were highly represented in the 12 °C bred, while those of Acinetobacter, Pseudochrobactrum, Enterococcus, Comamonas, and Leucobacter genera were significantly represented in the 16 °C bred group. Metagenomic revealed that several animal probiotics of the Enterococcus and Vagococcus genera were greatly enriched in the gut of larvae bred at 16 °C. Moreover, bacterial metabolic pathways including carbohydrate, lipid, amino acids, and cofactors and vitamins, were significantly increased, while organismal systems and human diseases was decreased in the 16 °C bred group. Transcriptomic analysis revealed that the upregulated differentially expressed genes in the 16 °C bred groups mainly participated in Autophagy-animal, AMPK signaling pathway, mTOR signaling pathway, Wnt signaling pathway, FoxO signaling pathway, Hippo signaling pathway at day 34 under 16 °C conditions, suggesting their significant role in the survival of BSFL. Taken together, these results shed lights on the role of intestinal microflora and gene pathways in the adaptation of BSF larvae to cold stress.

Production and characterization of Trichoderma asperellum chitinases and their use in synergy with Bacillus thuringiensis for lepidopteran control.

BACKGROUND: Despite their known negative effects on ecosystems and human health, synthetic pesticides are still largely used to control crop insect pests. Currently, the biopesticide market for insect biocontrol mainly relies on the entomopathogenic bacterium Bacillus thuringiensis (Bt). New biocontrol tools for crop protection might derive from fungi, in particular from Trichoderma spp., which are known producers of chitinases and other bioactive compounds able to negatively affect insect survival. RESULTS: In this study, we first developed an environmentally sustainable production process for obtaining chitinases from Trichoderma asperellum ICC012. Then, we investigated the biological effects of this chitinase preparation - alone or in combination with a Bt-based product - when orally administered to two lepidopteran species. Our results demonstrate that T. asperellum efficiently produces a multi-enzymatic cocktail able to alter the chitin microfibril network of the insect peritrophic matrix, resulting in delayed development and larval death. The co-administration of T. asperellum chitinases and sublethal concentrations of Bt toxins increased larval mortality. This synergistic effect was likely due to the higher amount of Bt toxins that passed the damaged peritrophic matrix and reached the target receptors on the midgut cells of chitinase-treated insects. CONCLUSION: Our findings may contribute to the development of an integrated pest management technology based on fungal chitinases that increase the efficacy of Bt-based products, mitigating the risk of Bt-resistance development. © 2024 Society of Chemical Industry.

Anatomical changes of Tenebrio molitor and Tribolium castaneum during complete metamorphosis.

In holometabolous insects, extensive reorganisation of tissues and cells occurs at the pupal stage. The remodelling of the external exoskeleton and internal organs that intervenes during metamorphosis has been traditionally studied in many insect species based on histological or ultrastructural methods. This study demonstrates the use of synchrotron X-ray phase-contrast micro-computed tomography as a powerful, non-destructive tool for in situ morphological observation of anatomical structures at the pupal stage in two Tenebrionid beetles, i.e. Tribolium castaneum and Tenebrio molitor, known as important pests, as well as emerging and promising models in experimental biology. Virtual sections and three-dimensional reconstructions were performed on both males and females at early, intermediate, and late pupal stage. The dataset allowed us to observe the remodelling of the gut and nervous system as well as the shaping of the female and male reproductive system at different pupal ages in both mealworm and red flour beetles. Moreover, we observed that the timing and duration pattern of organ development varied between the species analysed, likely related to the species-specific adaptations of the pre-imaginal stages to environmental conditions, which ultimately affect their life cycle. This research provides new knowledge on the morphological modifications that occur during the pupal stage of holometabolous insects and provides a baseline set of information on beetle metamorphosis that may support future research in forensics, physiology, and ecology as well as an image atlas for educational purposes.

Environmental impact scenarios of organic fraction municipal solid waste treatment with Black Soldier Fly larvae based on a life cycle assessment.

Biowaste treatment with Black Soldier Fly (BSF) larvae is an alternative option for organic waste valorization. Its environmental impacts should be assessed and compared with conventional treatment options. The research aims to evaluate the treatment of organic fraction of municipal solid waste (OFMSW) with BSF larvae through a life cycle assessment (LCA). This study employed data inventories from literature and aimed to provide a wide range of production parameter values to identify the potentialities of BSF treatment in the best-case and worst-case scenarios. The SimaPro9, the database Ecoinvent3.5, and the impact assessment method IMPACT 2002+ have been employed for the analysis. A sensitivity analysis of relevant parameters was conducted, considering the avoided impacts that can be obtained thanks to the exploitation of larvae proteins for bioplastics or fishmeal production. Research findings highlight six main environmental impact indicators: respiratory inorganics (kg PM2.5-eq), ozone layer depletion (kg CFC-11-eq), terrestrial ecotoxicity (kg TEG soil), land occupation (m2 organic arable), global warming (kg CO2-eq), and non-renewable energy (MJ primary). The most relevant process generating impacts is BSF breeding, followed by boiling, storage, and OFMSW treatment. The environmental performance is better when the conventional fishmeal substituted, thanks to BSF larvae production, is made from areas 10,000 km far, implementing a 100% renewable energy scenario, reducing the energy consumption by 50%, increasing the lifespan of the equipment to 15 years, and products are employed locally. The current study represents the first attempt to evaluate the global higher or lower environmental impact scenario related to OFMSW treatment through BSF larvae.

Bacterial biota composition in gut regions of black soldier fly larvae reared on industrial residual streams: revealing community dynamics along its intestinal tract.

Some insect species have gained attention as efficient bioconverters of low-value organic substrates (i.e., residual streams) into high-value biomass. Black soldier fly (BSF) (Hermetia illucens) larvae are particularly interesting for bioconversion due to their ability to grow on a wide range of substrates, including low-value industrial residual streams. This is in part due to the plasticity of the gut microbiota of polyphagous insects, like BSF. Gut microbiota composition varies depending on rearing substrates, via a mechanism that might support the recruitment of microorganisms that facilitate digestion of a specific substrate. At the same time, specific microbial genera do persist on different substrates via unknown mechanisms. This study aimed to offer insights on this microbial plasticity by investigating how the composition of the bacterial community present in the gut of BSF larvae responds to two industrial residual streams: swill (a mixture of catering and supermarket leftovers) and distiller's dried grains with solubles. The bacterial biota composition of substrates, whole larvae at the beginning of the rearing period and at harvest, rearing residues, and larval gut regions were investigated through 16S rRNA gene sequencing. It was observed that both substrate and insect development influenced the bacterial composition of the whole larvae. Zooming in on the gut regions, there was a clear shift in community composition from a higher to a lower diversity between the anterior/middle midgut and the posterior midgut/hindgut, indicating a selective pressure occurring in the middle midgut region. Additionally, the abundance of the bacterial biota was always high in the hindgut, while its diversity was relatively low. Even more, the bacterial community in the hindgut was found to be relatively more conserved over the different substrates, harboring members of the BSF core microbiota. We postulate a potential role of the hindgut as a reservoir for insect-associated microbes. This warrants further research on that underexplored region of the intestinal tract. Overall, these findings contribute to our understanding of the bacterial biota structure and dynamics along the intestinal tract, which can aid microbiome engineering efforts to enhance larval performance on (industrial) residual streams.

Plastics shape the black soldier fly larvae gut microbiome and select for biodegrading functions.

BACKGROUND: In the last few years, considerable attention has been focused on the plastic-degrading capability of insects and their gut microbiota in order to develop novel, effective, and green strategies for plastic waste management. Although many analyses based on 16S rRNA gene sequencing are available, an in-depth analysis of the insect gut microbiome to identify genes with plastic-degrading potential is still lacking. RESULTS: In the present work, we aim to fill this gap using Black Soldier Fly (BSF) as insect model. BSF larvae have proven capability to efficiently bioconvert a wide variety of organic wastes but, surprisingly, have never been considered for plastic degradation. BSF larvae were reared on two widely used plastic polymers and shotgun metagenomics was exploited to evaluate if and how plastic-containing diets affect composition and functions of the gut microbial community. The high-definition picture of the BSF gut microbiome gave access for the first time to the genomes of culturable and unculturable microorganisms in the gut of insects reared on plastics and revealed that (i) plastics significantly shaped bacterial composition at species and strain level, and (ii) functions that trigger the degradation of the polymer chains, i.e., DyP-type peroxidases, multicopper oxidases, and alkane monooxygenases, were highly enriched in the metagenomes upon exposure to plastics, consistently with the evidences obtained by scanning electron microscopy and 1H nuclear magnetic resonance analyses on plastics. CONCLUSIONS: In addition to highlighting that the astonishing plasticity of the microbiota composition of BSF larvae is associated with functional shifts in the insect microbiome, the present work sets the stage for exploiting BSF larvae as "bioincubators" to isolate microbial strains and enzymes for the development of innovative plastic biodegradation strategies. However, most importantly, the larvae constitute a source of enzymes to be evolved and valorized by pioneering synthetic biology approaches. Video Abstract.

Combined effect of a neonicotinoid insecticide and a fungicide on honeybee gut epithelium and microbiota, adult survival, colony strength and foraging preferences.

Fungicides, insecticides and herbicides are widely used in agriculture to counteract pathogens and pests. Several of these molecules are toxic to non-target organisms such as pollinators and their lethal dose can be lowered if applied as a mixture. They can cause large and unpredictable problems, spanning from behavioural changes to alterations in the gut. The present work aimed at understanding the synergistic effects on honeybees of a combined in-hive exposure to sub-lethal doses of the insecticide thiacloprid and the fungicide penconazole. A multidisciplinary approach was used: honeybee mortality upon exposure was initially tested in cage, and the colonies development monitored. Morphological and ultrastructural analyses via light and transmission electron microscopy were carried out on the gut of larvae and forager honeybees. Moreover, the main pollen foraging sources and the fungal gut microbiota were studied using Next Generation Sequencing; the gut core bacterial taxa were quantified via qPCR. The mortality test showed a negative effect on honeybee survival when exposed to agrochemicals and their mixture in cage but not confirmed at colony level. Microscopy analyses on the gut epithelium indicated no appreciable morphological changes in larvae, newly emerged and forager honeybees exposed in field to the agrochemicals. Nevertheless, the gut microbial profile showed a reduction of Bombilactobacillus and an increase of Lactobacillus and total fungi upon mixture application. Finally, we highlighted for the first time a significant honeybee diet change after pesticide exposure: penconazole, alone or in mixture, significantly altered the pollen foraging preference, with honeybees preferring Hedera pollen. Overall, our in-hive results showed no severe effects upon administration of sublethal doses of thiacloprid and penconazole but indicate a change in honeybees foraging preference. A possible explanation can be that the different nutritional profile of the pollen may offer better recovery chances to honeybees.

Ingestion and excretion dynamics of microplastics by black soldier fly larvae and correlation with mouth opening size.

Black soldier fly (BSF) larvae (Hermetia illucens) are voracious feeders that can be reared on food waste streams originating from the food industry and retailers. Because these food waste streams are automatically being unpacked in substantial amounts, they can contain microplastics, potentially jeopardising the larvae's chemical safety when applied as compound feed ingredients. During this study, the dynamics of ingestion and excretion of microplastics by BSF larvae reared on substrates containing different contents (wMP = 0.00, 0.01, 0.10, 0.50, 1.00, 3.00%) of fluorescent blue-labelled microplastics (median size, Dv(50) = 61.5 µm) were monitored. To correlate the particle size with their uptake, larval mouth opening dimensions were measured during the rearing process. In conclusion, it appeared that ingestion of microplastics by BSF larvae depends on initial particle load, mouth size, and consequently also age. The larvae took up between 131 (wMP = 0.01%) and 4866 (wMP = 3.00%) particles leading to bioaccumulation factors (BAF) between 0.12 (wMP = 3.00%) and 1.07 (wMP = 0.01%). Larvae also appeared to excrete the microplastics, lowering the BAFs to values between 0.01 (wMP = 3.00%) and 0.54 (wMP = 0.01%).

A soil fungus confers plant resistance against a phytophagous insect by disrupting the symbiotic role of its gut microbiota.

Plants generate energy flows through natural food webs, driven by competition for resources among organisms, which are part of a complex network of multitrophic interactions. Here, we demonstrate that the interaction between tomato plants and a phytophagous insect is driven by a hidden interplay between their respective microbiotas. Tomato plants colonized by the soil fungus Trichoderma afroharzianum, a beneficial microorganism widely used in agriculture as a biocontrol agent, negatively affects the development and survival of the lepidopteran pest Spodoptera littoralis by altering the larval gut microbiota and its nutritional support to the host. Indeed, experiments aimed to restore the functional microbial community in the gut allow a complete rescue. Our results shed light on a novel role played by a soil microorganism in the modulation of plant-insect interaction, setting the stage for a more comprehensive analysis of the impact that biocontrol agents may have on ecological sustainability of agricultural systems.

Morphofunctional characterization of hemocytes in black soldier fly larvae.

In insects, the cell-mediated immune response involves an active role of hemocytes in phagocytosis, nodulation, and encapsulation. Although these processes have been well documented in multiple species belonging to different insect orders, information concerning the immune response, particularly the hemocyte types and their specific function in the black soldier fly Hermetia illucens, is still limited. This is a serious gap in knowledge given the high economic relevance of H. illucens larvae in waste management strategies and considering that the saprophagous feeding habits of this dipteran species have likely shaped its immune system to efficiently respond to infections. The present study represents the first detailed characterization of black soldier fly hemocytes and provides new insights into the cell-mediated immune response of this insect. In particular, in addition to prohemocytes, we identified five hemocyte types that mount the immune response in the larva, and analyzed their behavior, role, and morphofunctional changes in response to bacterial infection and injection of chromatographic beads. Our results demonstrate that the circulating phagocytes in black soldier fly larvae are plasmatocytes. These cells also take part in nodulation and encapsulation with granulocytes and lamellocyte-like cells, developing a starting core for nodule/capsule formation to remove/encapsulate large bacterial aggregates/pathogens from the hemolymph, respectively. These processes are supported by the release of melanin precursors from crystal cells and likely by mobilizing nutrient reserves in newly circulating adipohemocytes, which could thus trophically support other hemocytes during the immune response. Finally, the regulation of the cell-mediated immune response by eicosanoids was investigated.

Identification of Host Molecules Involved in the Proliferation of Nucleopolyhedrovirus in Bombyx mori.

The Bombyx mori nucleopolyhedrovirus (BmNPV), a foodborne infectious virus, is the pathogen causing nuclear polyhedrosis and high lethality in the silkworm. In this study, we characterized the molecules involved in BmNPV-silkworm interaction by RNA sequencing of the fat body isolated from the virus-susceptible strain P50. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation showed that the upregulated differentially expressed genes (DEGs) were mainly involved in translation, signal transduction, folding, sorting, and degradation, as well as transport and catabolism, while the downregulated DEGs were predominantly enriched in the metabolism of carbohydrates, amino acids, and lipids at 72 h post BmNPV infection. Knockout of the upregulated somatomedin-B and thrombospondin type-1 domain-containing protein, probable allantoicase, trifunctional purine biosynthetic protein adenosine-3, and Psl and pyoverdine operon regulator inhibited the proliferation of BmNPV, while knockout of the downregulated clip domain serine protease 3 and carboxylesterase clade H, member 1 promoted it. The molecules herein identified provide a foundation for developing strategies and designing drugs against BmNPV.

Silkworm and Silk: Traditional and Innovative Applications.

The various subjects covered in the present Special Issue "Silkworm and Silk: Traditional and Innovative Applications" demonstrate how sericulture, a practice deeply rooted in human history, can act as a bridge to bring together an exceptionally wide range of scientific and technical expertise in both conventional topics and cutting-edge technologies [...].

A Bombyx mori Infection Model for Screening Antibiotics against Staphylococcus epidermidis.

The increasing number of microorganisms that are resistant to antibiotics is prompting the development of new antimicrobial compounds and strategies to fight bacterial infections. The use of insects to screen and test new drugs is increasingly considered a promising tool to accelerate the discovery phase and limit the use of mammalians. In this study, we used for the first time the silkworm, Bombyx mori, as an in vivo infection model to test the efficacy of three glycopeptide antibiotics (GPAs), against the nosocomial pathogen Staphylococcus epidermidis. To reproduce the human physiological temperature, the bacterial infection was performed at 37 °C and it was monitored over time by evaluating the survival rate of the larvae, as well the response of immunological markers (i.e., activity of hemocytes, activation of the prophenoloxidase system, and lysozyme activity). All the three GPAs tested (vancomycin, teicoplanin, and dalbavancin) were effective in curing infected larvae, significantly reducing their mortality and blocking the activation of the immune system. These results corroborate the use of this silkworm infection model for the in vivo studies of antimicrobial molecules active against staphylococci.

New value from food and industrial wastes - Bioaccumulation of omega-3 fatty acids from an oleaginous microbial biomass paired with a brewery by-product using black soldier fly (Hermetia illucens) larvae.

Research on bioconversion based on insects is intensifying as it addresses the problem of reducing and reusing food and industrial waste. To reach this goal, we need to find more means of pairing waste to insects. With this goal, brewers' spent grains (BSG) - a food waste of the brewing industry - paired with the oleaginous biomass of the thraustochytrid Schizochytrium limacinum cultivated on crude glycerol - a major waste of biodiesel production - were successfully used to grow Hermetia illucens larvae. Combining BSG and S. limacinum in the diet in an attempt to design the lipid profile of H. illucens larvae to contain a higher percentage of omega-3 fatty acids is novel. Insect larvae were grown on three different substrates: i) standard diet for Diptera (SD), ii) BSG, and iii) BSG + 10% S. limacinum biomass. The larvae and substrates were analyzed for fatty acid composition and larval growth was measured until 25% of insects reached the prepupal stage. Our data showed that including omega-3-rich S. limacinum biomass in the BSG substrate promoted an increase in larval weight compared to larvae fed on SD or BSG substrates. Furthermore, it was possible, albeit in a limited way, to incorporate omega-3 fatty acids, principally docosahexaenoic acid (DHA) from BSG + S. limacinum substrate containing 20% of DHA into the larval fat (7% DHA). However, H. illucens with this level of DHA may not be suitable if the aim is to get larvae with high omega-3 lipids to feed carnivorous fish.

Transcriptional and Post-Transcriptional Regulation of Autophagy.

Autophagy is a widely conserved process in eukaryotes that is involved in a series of physiological and pathological events, including development, immunity, neurodegenerative disease, and tumorigenesis. It is regulated by nutrient deprivation, energy stress, and other unfavorable conditions through multiple pathways. In general, autophagy is synergistically governed at the RNA and protein levels. The upstream transcription factors trigger or inhibit the expression of autophagy- or lysosome-related genes to facilitate or reduce autophagy. Moreover, a significant number of non-coding RNAs (microRNA, circRNA, and lncRNA) are reported to participate in autophagy regulation. Finally, post-transcriptional modifications, such as RNA methylation, play a key role in controlling autophagy occurrence. In this review, we summarize the progress on autophagy research regarding transcriptional regulation, which will provide the foundations and directions for future studies on this self-eating process.

Insights Into the Immune Response of the Black Soldier Fly Larvae to Bacteria.

In insects, a complex and effective immune system that can be rapidly activated by a plethora of stimuli has evolved. Although the main cellular and humoral mechanisms and their activation pathways are highly conserved across insects, the timing and the efficacy of triggered immune responses can differ among different species. In this scenario, an insect deserving particular attention is the black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae). Indeed, BSF larvae can be reared on a wide range of decaying organic substrates and, thanks to their high protein and lipid content, they represent a valuable source of macromolecules useful for different applications (e.g., production of feedstuff, bioplastics, and biodiesel), thus contributing to the development of circular economy supply chains for waste valorization. However, decaying substrates bring the larvae into contact with different potential pathogens that can challenge their health status and growth. Although these life strategies have presumably contributed to shape the evolution of a sophisticated and efficient immune system in this dipteran, knowledge about its functional features is still fragmentary. In the present study, we investigated the processes underpinning the immune response to bacteria in H. illucens larvae and characterized their reaction times. Our data demonstrate that the cellular and humoral responses in this insect show different kinetics: phagocytosis and encapsulation are rapidly triggered after the immune challenge, while the humoral components intervene later. Moreover, although both Gram-positive and Gram-negative bacteria are completely removed from the insect body within a few hours after injection, Gram-positive bacteria persist in the hemolymph longer than do Gram-negative bacteria. Finally, the activity of two key actors of the humoral response, i.e., lysozyme and phenoloxidase, show unusual dynamics as compared to other insects. This study represents the first detailed characterization of the immune response to bacteria of H. illucens larvae, expanding knowledge on the defense mechanisms of this insect among Diptera. This information is a prerequisite to manipulating the larval immune response by nutritional and environmental factors to increase resistance to pathogens and optimize health status during mass rearing.

Diversity of insect antimicrobial peptides and proteins - A functional perspective: A review.

The innate immune response of insects provides a robust line of defense against pathogenic microbes and eukaryotic parasites. It consists of two types of overlapping immune responses, named humoral and cellular, which share protective molecules and regulatory mechanisms that closely coordinate to prevent the spread and replication of pathogens within the compromised insect hemocoel. The major feature of the humoral part of the insect immune system involves the production and secretion of antimicrobial peptides from the fat body, which is considered analogous to adipose tissue and liver in vertebrates. Previous research has identified and characterized the nature of antimicrobial peptides that are directed against various targets during the different stages of infection. Here we review this information focusing mostly on the diversity and mode of action of these host defense components, and their critical contribution to maintaining host homeostasis. Extending this knowledge is paramount for understanding the evolution of innate immune function and the physiological balance required to provide sufficient protection to the host against external enemies while avoiding overactivation signaling events that would severely undermine physiological stability.