Preliminary Toxicity Evaluation of a Porphyrin Photosensitizer in an Alternative Preclinical Model.

In photodynamic therapy (PDT), a photosensitizer (PS) excited with a specific wavelength, and in the presence of oxygen, gives rise to photochemical reactions that lead to cell damage. Over the past few years, larval stages of the G. mellonella moth have proven to be an excellent alternative animal model for in vivo toxicity testing of novel compounds and virulence testing. In this article, we report a series of preliminary studies on G. mellonella larvae to evaluate the photoinduced stress response by a porphyrin (PS) (TPPOH). The tests performed evaluated PS toxicity on larvae and cytotoxicity on hemocytes, both in dark conditions and following PDT. Cellular uptake was also evaluated by fluorescence and flow cytometry. The results obtained demonstrate how the administration of PS and subsequent irradiation of larvae affects not only larvae survival rate, but also immune system cells. It was also possible to verify PS's uptake and uptake kinetics in hemocytes, observing a maximum peak at 8 h. Given the results obtained in these preliminary tests, G. mellonella appears to be a promising model for preclinical PS tests.

Optimization of Experimental Infection of the Animal Model Galleria mellonella Linnaeus 1758 (Lepidoptera: Pyralidae) with the Gram-Positive Bacterium Micrococcus luteus.

The aim of this work was to develop an experimental protocol for the infection of Galleria mellonella with Gram-positive bacteria. Some physiological characteristics of these insects are comparable to those of vertebrates, therefore allowing the replacement of mammals in the preclinical phases of drug development. G. mellonella Linnaeus 1758 (Lepidoptera: Pyralidae) is accepted as an alternative model for the study of infectious diseases. Since data on infection procedures with different bacterial strains are scarce and sometimes conflicting, also due to different and non-uniform protocols, we developed an experimental protocol that would allow for controlled and repeatable infections, using the Gram-positive bacterium GRAS (Generally Regarded As Safe) Micrococcus luteus. After analyzing the morphology and defining the growth rate of M. luteus, doses of between 101 and 106 CFU/larvae were administered to late-stage larvae. The survival rate of the larvae was monitored up to 7 days and the LD50 determined. The bacterial clearance capacity of the larvae after injection with 103 and 105 CFU/larvae was assessed by hemolymph bacterial load analysis. The results made it possible to define the growth curve of M. luteus correlated with the CFU count; based on the LD50 (103.8 CFU/larvae) calculated on the survival of G. mellonella, infections were carried out to evaluate the immune efficiency of the larvae in bacterial clearance. This protocol, standardized on G. mellonella larvae, could provide a functional tool to study the course of bacterial infections.

Entomopathogenic potential of bacteria associated with soil-borne nematodes and insect immune responses to their infection.

Soil-borne nematodes establish close associations with several bacterial species. Whether they confer benefits to their hosts has been investigated in only a few nematode-bacteria systems. Their ecological function, therefore, remains poorly understood. In this study, we isolated several bacterial species from rhabditid nematodes, molecularly identified them, evaluated their entomopathogenic potential on Galleria mellonella larvae, and measured immune responses of G. mellonella larvae to their infection. Bacteria were isolated from Acrobeloides sp., A. bodenheimeri, Heterorhabditis bacteriophora, Oscheius tipulae, and Pristionchus maupasi nematodes. They were identified as Acinetobacter sp., Alcaligenes sp., Bacillus cereus, Enterobacter sp., Kaistia sp., Lysinibacillus fusiformis, Morganella morganii subsp. morganii, Klebsiella quasipneumoniae subsp. quasipneumoniae, and Pseudomonas aeruginosa. All bacterial strains were found to be highly entomopathogenic as they killed at least 53.33% G. mellonella larvae within 72h post-infection, at a dose of 106 CFU/larvae. Among them, Lysinibacillus fusiformis, Enterobacter sp., Acinetobacter sp., and K. quasipneumoniae subsp. quasipneumoniae were the most entomopathogenic bacteria. Insects strongly responded to bacterial infection. However, their responses were apparently little effective to counteract bacterial infection. Our study, therefore, shows that bacteria associated with soil-borne nematodes have entomopathogenic capacities. From an applied perspective, our study motivates more research to determine the potential of these bacterial strains as biocontrol agents in environmentally friendly and sustainable agriculture.

The Influence of Daily Temperature Fluctuation on the Efficacy of Bioinsecticides on Spotted Wing Drosophila Larvae.

Global climate change is allowing the invasion of insect pests into new areas without natural competitors and/or predators. The dipteran Drosophila suzukii has invaded both the Americas and Europe, becoming a serious problem for fruit crops. Control methods for this pest are still based on the use of pesticides, but less invasive and more sustainable methods, such as biocontrol, are needed. Variations in environmental conditions can affect the efficacy of bioinsecticides influencing their behavior and physiology besides that of the target insects. In this work, we developed a system that simulates the daily temperature fluctuations (DTFs) detected in the environment, with the aim of studying the influence of temperature on biocontrol processes. We investigated the effects of DTFs on the efficacy of four bioinsecticides. Results showed that DTFs modify the efficacy of some entomopathogens while they are ineffective on others. Specifically, the bacterium Bacillus thuringiensis is the most effective bioinsecticide under all conditions tested, i.e., low DTF (11−22 °C) and high DTF (17−33 °C) compared to constant temperature (25 °C). In contrast, nematodes are more sensitive to changes in temperature: Steinernema carpocapsae loses efficacy at low DTF, while Steinernema feltiae and Heterorhabditis bacteriophora are not effective in controlling the target dipteran. This work provides a basis for reviewing biological control methods against invasive species in the current context of climate change.

The Silkworm as a Source of Natural Antimicrobial Preparations: Efficacy on Various Bacterial Strains.

The global spread of multi-resistant pathogens responsible for infections, which cannot be treated with existing drugs such as antibiotics, is of particular concern. Antibiotics are becoming increasingly ineffective and drug resistance is leading to more difficult-to-treat infections; therefore, new bioactive compounds with antimicrobial activity are needed and new alternative sources should be found. Antimicrobial peptides (AMPs) are synthesized by processes typical of the innate immune system and are present in almost all organisms. Insects are extremely resistant to bacterial infections as they can produce a wide range of AMPs, providing an effective first line of defense. The AMPs produced by insects therefore represent a possible source of natural antimicrobial molecules. In this paper, the possibility of using plasma preparations from silkworm (Bombyx mori) larvae as a source of antimicrobials was evaluated. After simple purification steps, insect plasma was analyzed and tested on different Gram-positive and Gram-negative bacterial strains. The results obtained are encouraging as the assays on Escherichia coli and Enterobacter cloacae showed significant decrease in the growth of these Gram-negative bacteria. Similar results were obtained on Gram-positive bacteria, such as Micrococcus luteus and Bacillus subtilis, which showed strong susceptibility to the silkworm AMPs pool. In contrast, Staphylococcus aureus displayed high resistance to Bombyx mori plasma. Finally, the tested plasma formulations were assessed for possible storage not only at 4 °C, but also above room temperature. In conclusion, partially purified plasma from silkworm could be a promising source of AMPs which could be used in formulations for topical applications, without additional and expensive purification steps.

Drosophila suzukii Susceptibility to the Oral Administration of Bacillus thuringiensis, Xenorhabdus nematophila and Its Secondary Metabolites.

Drosophila suzukii, Spotted Wing Drosophila (SWD), is a serious economic issue for thin-skinned fruit farmers. The invasion of this dipteran is mainly counteracted by chemical control methods; however, it would be desirable to replace them with biological control. All assays were performed with Bacillus thuringiensis (Bt), Xenorhabdus nematophila (Xn), and Xn secretions, administered orally in single or combination, then larval lethality was assessed at different times. Gut damage caused by Bt and the influence on Xn into the hemocoelic cavity was also evaluated. In addition, the hemolymph cell population was analyzed after treatments. The data obtained show that the combined use of Bt plus Xn secretions on larvae, compared to single administration of bacteria, significantly improved the efficacy and reduced the time of treatments. The results confirm the destructive action of Bt on the gut of SWD larvae, and that Bt-induced alteration promotes the passage of Xn to the hemocoel cavity. Furthermore, hemocytes decrease after bioinsecticides treatments. Our study demonstrates that combining bioinsecticides can improve the efficacy of biocontrol and such combinations should be tested in greenhouse and in field in the near future.

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.

When Appearance Misleads: The Role of the Entomopathogen Surface in the Relationship with Its Host.

Currently, potentially harmful insects are controlled mainly by chemical synthetic insecticides, but environmental emergencies strongly require less invasive control techniques. The use of biological insecticides in the form of entomopathogenic organisms is undoubtedly a fundamental resource for the biological control of insect pests in the future. These infectious agents and endogenous parasites generally act by profoundly altering the host's physiology to death, but their success is closely related to the neutralization of the target insect's immune response. In general, entomopathogen parasites, entomopathogenic bacteria, and fungi can counteract immune processes through the effects of secretion/excretion products that interfere with and damage the cells and molecules typical of innate immunity. However, these effects are observed in the later stages of infection, whereas the risk of being recognized and neutralized occurs very early after penetration and involves the pathogen surface components and molecular architecture; therefore, their role becomes crucial, particularly in the earliest pathogenesis. In this review, we analyze the evasion/interference strategies that entomopathogens such as the bacterium Bacillus thuringiensis, fungi, nematocomplexes, and wasps implement in the initial stages of infection, i.e., the phases during which body or cell surfaces play a key role in the interaction with the host receptors responsible for the immunological discrimination between self and non-self. In this regard, these organisms demonstrate evasive abilities ascribed to their body surface and cell wall; it appears that the key process of these mechanisms is the capability to modify the surface, converting it into an immunocompatible structure, or interaction that is more or less specific to host factors.

Immune Response of Drosophila suzukii Larvae to Infection with the Nematobacterial Complex Steinernema carpocapsae-Xenorhabdus nematophila.

Entomopathogenic nematodes have been proposed as biological agents for the control of Drosophila suzukii, an invasive pest of small-stone and soft-skinned fruits. Larvae of the fly are susceptible to Steinernema carpocapsae infection but the reaction of immune defenses of the host are unknown. To determine the immune response, larvae were infected with S. carpocapsae and Xenorhabdus nematophila to evaluate the effector mechanisms of both humoral and cellular processes. The symbiont bacteria presented an inhibitory effect on the phenoloxidase cascade with a low level of melanization. Besides, X. nematophila activated the synthesis of putative antimicrobial peptides on the hemolymph of infected larvae. However, those peptides presented a lower antimicrobial activity compared to hemolymph from larvae infected with non-symbiont bacteria. Xenorhabdus nematophila avoided also the phagocytosis response of hemocytes. During in vitro and in vivo assays, S. carpocapsae was not encapsulated by cells, unless the cuticle was damaged with a lipase-treatment. Hemocyte counts confirmed differentiation of lamellocytes in the early phase of infection despite the unrecognition of the nematodes. Both X. nematophila and S. carpocapsae avoided the cellular defenses of D. suzukii larvae and depressed the humoral response. These results confirmed the potential of entomopathogenic nematodes to control D. suzukii.

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.

Nematobacterial Complexes and Insect Hosts: Different Weapons for the Same War.

Entomopathogenic nematodes (EPNs) are widely used as biological control agents against insect pests, the efficacy of these organisms strongly depends on the balance between the parasitic strategies and the immune response of the host. This review summarizes roles and relationships between insect hosts and two well-known EPN species, Steinernema feltiae and Steinernema carpocapsae and outlines the main mechanisms of immune recognition and defense of insects. Analyzing information and findings about these EPNs, it is clear that these two species use shared immunosuppression strategies, mainly mediated by their symbiotic bacteria, but there are differences in both the mechanism of evasion and interference of the two nematodes with the insect host immune pathways. Based on published data, S. feltiae takes advantage of the cross reaction between its body surface and some host functional proteins, to inhibit defensive processes; otherwise, secretion/excretion products from S. carpocapsae seem to be the main nematode components responsible for the host immunosuppression.

Surface protein components from entomopathogenic nematodes and their symbiotic bacteria: effects on immune responses of the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae).

BACKGROUND: Steinernema carpocapsae is a nematocomplex widely used as an alternative to chemicals for the biological control of insect pests; this nematode is symbiotically associated with the bacterium Xenorhabdus nematophila and both contribute to host death. The architecture and functions of structures and molecular components of the surface of nematodes and their symbiont bacteria are integral to early interactions with their hosts; thus, we assessed the role of protein pools isolated from the surface of S. carpocapsae and from phase I X. nematophila against Galleria mellonella. RESULTS: Using high-salt treatments, we isolated the surface proteins and assayed them on G. mellonella haemocytes; haemocyte viability and phagocytic activity were investigated in the presence of surface proteins from nematodes or bacteria. Proteins from live S. carpocapsae possessed mild cytotoxicity on the haemocytes, whereas those from live X. nematophila markedly affected the host cells' viability. Bacterial proteins inhibited phagocytic activity, although they strongly triggered the host proPO (prophenoloxidase-phenoloxidase) system. CONCLUSION: Nematocomplex surface compounds play a key role in immunoevasion/depression of insect hosts, causing a severe physiological disorder. Natural compounds newly identified as active against pests could improve the pest management of species potentially harmful to plants in urban green spaces and agriculture. © 2018 Society of Chemical Industry.