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.

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.

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.

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.

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.

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.

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.

A Silkworm Infection Model for In Vivo Study of Glycopeptide Antibiotics.

Glycopeptide antibiotics (GPAs) are drugs of last resort for treating infections by Gram-positive bacteria. They inhibit bacterial cell wall assembly by binding to the d-Ala-d-Ala terminus of peptidoglycan precursors, leading to cell lysis. Vancomycin and teicoplanin are first generation GPAs, while dalbavancin is one of the few, recently approved, second generation GPAs. In this paper, we developed an in vivo insect model to compare, for the first time, the efficacy of these three GPAs in curing Staphylococcus aureus infection. Differently from previous reports, Bombyx mori larvae were reared at 37 °C, and the course of infection was monitored, following not only larval survival, but also bacterial load in the insect body, hemocyte activity, phenoloxidase activity, and antimicrobial peptide expression. We demonstrated that the injection of S. aureus into the hemolymph of B. mori larvae led to a marked reduction of their survival rate within 24-48 hours. GPAs were not toxic to the larvae and cured S. aureus infection. Dalbavancin was more effective than first generation GPAs. Due to its great advantages (i.e., easy and safe handling, low rearing costs, low antibiotic amount needed for the tests, no restrictions imposed by ethical and regulatory issues), this silkworm infection model could be introduced in preclinical phases-prior to the use of mice-accelerating the discovery/development rate of novel GPAs.

Methods for Monitoring Autophagy in Silkworm Organs.

In holometabolous insects, various larval organs are remodeled by autophagy during metamorphosis. Although moths and butterflies are among the first animal models in which this self-eating process was described, only in recent years autophagy has been analyzed in detail in these insects. In particular, the silkworm Bombyx mori, which represents a well-studied model among Lepidoptera, provides a wide repertoire of cellular and molecular tools useful for studying the occurrence of autophagy and for evaluating its role in postembryonic development. Here, we describe some morphological, biochemical, and molecular methods to monitor autophagy in silkworm organs.

Metagenome-Sourced Microbial Chitinases as Potential Insecticide Proteins.

Microbial chitinases are gaining interest as promising candidates for controlling plant pests. These enzymes can be used directly as biocontrol agents as well as in combination with chemical pesticides or other biopesticides, reducing their environmental impact and/or enhancing their efficacy. Chitinolytic enzymes can target two different structures in insects: the cuticle and the peritrophic matrix (PM). PM, formed by chitin fibrils connected to glycoproteins and proteoglycans, represents a physical barrier that plays an essential role in midgut physiology and insect digestion, and protects the absorptive midgut epithelium from food abrasion or pathogen infections. In this paper, we investigate how two recently discovered metagenome-sourced chitinases (Chi18H8 and 53D1) affect, in vitro and in vivo, the PM integrity of Bombyx mori, a model system among Lepidoptera. The two chitinases were produced in Escherichia coli or, alternatively, in the unconventional - but more environmentally acceptable - Streptomyces coelicolor. Although both the proteins dramatically altered the structure of B. mori PM in vitro, when administered orally only 53D1 caused adverse and marked effects on larval growth and development, inducing mortality and reducing pupal weight. These in vivo results demonstrate that 53D1 is a promising candidate as insecticide protein.

Enhanced Silkworm Cecropin B Antimicrobial Activity against Pseudomonas aeruginosa from Single Amino Acid Variation.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen causing severe infections in hospitalized and immunosuppressed patients, particularly individuals affected by cystic fibrosis. Several clinically isolated P. aeruginosa strains were found to be resistant to three or more antimicrobial classes indicating the importance of identifying new antimicrobials active against this pathogen. Here, we characterized the antimicrobial activity and the action mechanisms against P. aeruginosa of two natural isoforms of the antimicrobial peptide cecropin B, both isolated from the silkworm Bombyx mori. These cecropin B isoforms differ in a single amino acid substitution within the active portion of the peptide, so that the glutamic acid of the E53 CecB variant is replaced by a glutamine in the Q53 CecB isoform. Both peptides showed a high antimicrobial and membranolytic activity against P. aeruginosa, with Q53 CecB displaying greater activity compared with the E53 CecB isoform. Biophysical analyses, live-cell NMR, and molecular-dynamic-simulation studies indicated that both peptides might act as membrane-interacting elements, which can disrupt outer-membrane organization, facilitating their translocation toward the inner membrane of the bacterial cell. Our data also suggest that the amino acid variation of the Q53 CecB isoform represents a critical factor in stabilizing the hydrophobic segment that interacts with the bacterial membrane, determining the highest antimicrobial activity of the whole peptide. Its high stability to pH and temperature variations, tolerance to high salt concentrations, and low toxicity against human cells make Q53 CecB a promising candidate in the development of CecB-derived compounds against P. aeruginosa.

Timing of autophagy and apoptosis during posterior silk gland degeneration in Bombyx mori.

Over the years, the silkworm, Bombyx mori, has been manipulated by means of chemical and genetic approaches to improve silk production both quantitatively and qualitatively. The silk is produced by the silk gland, which degenerates quickly once the larva has finished spinning the cocoon. Thus, interfering with this degeneration process could help develop new technologies aimed at ameliorating silk yield. To this end, in this work we studied the cell death processes that lead to the demise of the posterior silk gland of B. mori, directing in particular our attention to autophagy and apoptosis. We focused on this portion of the gland because it produces fibroin, the main component of the silk thread. By using multiple markers, we provide a morphological, biochemical and molecular characterization of the apoptotic and autophagic processes and define their timing in this biological setting. Our data demonstrate that the activation of both autophagy and apoptosis is preceded by a transcriptional rise in key regulatory genes. Moreover, while autophagy is maintained active for several days and progressively digests silk gland cells, apoptosis is only switched on at a very late stage of silk gland demise.

Midgut epithelium in molting silkworm: A fine balance among cell growth, differentiation, and survival.

The midgut of insects has attracted great attention as a system for studying intestinal stem cells (ISCs) as well as cell death-related processes, such as apoptosis and autophagy. Among insects, Lepidoptera represent a good model to analyze these cells and processes. In particular, larva-larva molting is an interesting developmental phase since the larva must deal with nutrient starvation and its organs are subjected to rearrangements due to proliferation and differentiation events. Several studies have analyzed ISCs in vitro and characterized key factors involved in their division and differentiation during molt. However, in vivo studies performed during larva-larva transition on these cells, and on the whole midgut epithelium, are fragmentary. In the present study, we analyzed the larval midgut epithelium of the silkworm, Bombyx mori, during larva-larva molting, focusing our attention on ISCs. Moreover, we investigated the metabolic changes that occur in the epithelium and evaluated the intervention of autophagy. Our data on ISCs proliferation and differentiation, autophagy activation, and metabolic and functional activities of the midgut cells shed light on the complexity of this organ during the molting phase.