A Case of Myiasis Caused by Cordylobia anthropophaga (Diptera: Calliphoridae) in an Italian Traveler Returning from Senegal.

PURPOSE: Myiases are infestations of human and animal tissues by fly larvae. These conditions are widespread in tropical countries and travelers in those areas are at risk of becoming infested. Although Cordylobia anthropophaga (Blanchard & Berenger-Feraud, 1872) is one of the most common myiasis-causing species, few high-quality images and molecular sequences are available for this fly. We present a case of C. anthropophaga infestation in an Italian patient returning from Senegal, with the aim of increasing both visual and molecular data for this species. METHODS: After removal, the larva was determined following standardized morphological keys and photographed under a digital microscope. Molecular characterization of the Cytochrome c oxidase subunit I (COI) was performed using universal primers. RESULTS: The general appearance, the structural organization of the cephalic region, of the cephaloskeleton, and of the posterior tracheal spiracles suggested that the causative agent of the myiasis was a third instar larva of C. anthropophaga. The morphological data are further supported by the molecular data: the COI sequence showed high levels of identity with the already published verified COI sequences of C. anthropophaga. CONCLUSION: We provide high-quality morphological and molecular data useful for the identification of larvae of C. anthropophaga. We highlight that myiasis might be common in Senegal and better data about its prevalence in travelers and in the endemic countries are needed to understand the burden of this condition.

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.

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.

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.

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 [...].

The Early Season Community of Flower-Visiting Arthropods in a High-Altitude Alpine Environment.

In mountain ecosystems, climate change can cause spatiotemporal shifts, impacting the composition of communities and altering fundamental biotic interactions, such as those involving flower-visiting arthropods. On of the main problems in assessing the effects of climate change on arthropods in these environments is the lack of baseline data. In particular, the arthropod communities on early flowering high-altitude plants are poorly investigated, although the early season is a critical moment for possible mismatches. In this study, we characterised the flower-visiting arthropod community on the early flowering high-altitude Alpine plant, Androsace brevis (Primulaceae). In addition, we tested the effect of abiotic factors (temperature and wind speed) and other variables (time, i.e., hour of the day, and number of flowers per plant) on the occurrence, abundance, and diversity of this community. A. brevis is a vulnerable endemic species growing in the Central Alps above 2000 m asl and flowering for a very short period immediately after snowmelt, thus representing a possible focal plant for arthropods in this particular moment of the season. Diptera and Hymenoptera were the main flower visitors, and three major features of the community emerged: an evident predominance of anthomyiid flies among Diptera, a rare presence of bees, and a relevant share of parasitoid wasps. Temperature and time (hour of the day), but not wind speed and number of flowers per plant, affected the flower visitors' activity. Our study contributes to (1) defining the composition of high-altitude Alpine flower-visiting arthropod communities in the early season, (2) establishing how these communities are affected by environmental variables, and (3) setting the stage for future evaluation of climate change effects on flower-visiting arthropods in high-altitude environments in the early season.

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.

Mechanical Processing of Hermetia illucens Larvae and Bombyx mori Pupae Produces Oils with Antimicrobial Activity.

The aim of this work was to develop processing methods that safeguard the quality and antimicrobial properties of H. illucens and B. mori oils. We adopted a vegetable diet for both insects: leftover vegetables and fruit for H. illucens and mulberry leaves for B. mori. First, alternative techniques to obtain a good oil extraction yield from the dried biomass of H. illucens larvae were tested. Traditional pressing resulted to be the best system to maximize the oil yield and it was successfully applied to B. mori pupae. Oil quality resulted comparable to that obtained with other extraction methods described in the literature. In the case of B. mori pupae, different treatments and preservation periods were investigated to evaluate their influence on the oil composition and quality. Interestingly, agar diffusion assays demonstrated the sensitivity of Gram-positive Bacillus subtilis and Staphylococcus aureus to H. illucens and B. mori derived oils, whereas the growth of Gram-negative Pseudomonas aeruginosa and Escherichia coli was not affected. This study confirms that fat and other active compounds of the oil extracted by hot pressing could represent effective antimicrobials against bacteria, a relevant result if we consider that they are by-products of the protein extraction process in the feed industry.

Manual Sampling and Video Observations: An Integrated Approach to Studying Flower-Visiting Arthropods in High-Mountain Environments.

Despite the rising interest in biotic interactions in mountain ecosystems, little is known about high-altitude flower-visiting arthropods. In particular, since the research in these environment can be limited or undermined by harsh conditions and logistical difficulties, it is mandatory to develop effective approaches that maximize possibilities to gather high-quality data. Here we compared two different methods, manual sampling and video observations, to investigate the interactions between the high-mountain arthropod community and flowers of Androsace brevis (Primulaceae), a vulnerable endemic alpine species with a short flowering period occurring in early season. We manually sampled flower-visiting arthropods according to the timed-observations method and recorded their activity on video. We assessed differences and effectiveness of the two approaches to estimate flower-visiting arthropod diversity and to identify potential taxa involved in A. brevis pollination. Both methods proved to be effective and comparable in describing the diversity of flower visitors at a high taxonomic level. However, with manual sampling we were able to obtain a fine taxonomic resolution for sampled arthropods and to evaluate which taxa actually carry A. brevis pollen, while video observations were less invasive and allowed us to assess arthropod behavior and to spot rare taxa. By combining the data obtained with these two approaches we could accurately identify flower-visiting arthropods, characterize their behavior, and hypothesize a role of Hymenoptera Apoidea and Diptera Brachycera in A. brevis pollination. Therefore, we propose integrating the two approaches as a powerful instrument to unravel interactions between flowering plants and associated fauna that can provide crucial information for the conservation of vulnerable environments such as high-mountain ecosystems.

An in-depth description of head morphology and mouthparts in larvae of the black soldier fly Hermetia illucens.

The larvae of the black soldier fly (BSF) Hermetia illucens are increasingly being used for waste management purposes given their ability to grow on a wide range of organic decaying materials. Although significant efforts have been spent to improve the mass rearing of BSF larvae on specific substrates and their bioconversion capability, little is known about the biology of this insect, especially with regards to the digestive system. In this study, we analyzed the morphology of the head and buccal apparatus of H. illucens larvae by using optical and scanning electron microscopy, evaluating the different mouthparts and their modifications during larval development. Our analysis showed that the larval head of H. illucens presents similarities to those of campodeiform insect larvae, whereas the mandibular-maxillary complex represents a food intake solution typical of Stratiomyidae that enables BSF larvae to ingest semiliquid food. The mouthparts resemble a "tunnel boring machine", where the hypopharynx separates finer organic particles from coarser and inorganic ones.

Black Soldier Fly Larvae Adapt to Different Food Substrates through Morphological and Functional Responses of the Midgut.

Modulation of nutrient digestion and absorption is one of the post-ingestion mechanisms that guarantees the best exploitation of food resources, even when they are nutritionally poor or unbalanced, and plays a pivotal role in generalist feeders, which experience an extreme variability in diet composition. Among insects, the larvae of black soldier fly (BSF), Hermetia illucens, can grow on a wide range of feeding substrates with different nutrient content, suggesting that they can set in motion post-ingestion processes to match their nutritional requirements. In the present study we address this issue by investigating how the BSF larval midgut adapts to diets with different nutrient content. Two rearing substrates were compared: a nutritionally balanced diet for dipteran larvae and a nutritionally poor diet that mimics fruit and vegetable waste. Our data show that larval growth performance is only moderately affected by the nutritionally poor diet, while differences in the activity of digestive enzymes, midgut cell morphology, and accumulation of long-term storage molecules can be observed, indicating that diet-dependent adaptation processes in the midgut ensure the exploitation of poor substrates. Midgut transcriptome analysis of larvae reared on the two substrates showed that genes with important functions in digestion and absorption are differentially expressed, confirming the adaptability of this organ.

Estimating black soldier fly larvae biowaste conversion performance by simulation of midgut digestion.

Black soldier fly larvae treatment is an emerging technology for the conversion of biowaste into potentially more sustainable and marketable high-value products, according to circular economy principles. Unknown or variable performance for different biowastes is currently one challenge that prohibits the global technology up-scaling. This study describes simulated midgut digestion for black soldier fly larvae to estimate biowaste conversion performance. Before simulation, the unknown biowaste residence time in the three midgut regions was determined on three diets varying in protein and non-fiber carbohydrate content. For the static in vitro model, diet residence times of 15 min, 45 min, and 90 min were used for the anterior, middle, and posterior midgut region, respectively. The model was validated by comparing the ranking of diets based on in vitro digestion products to the ranking found in in vivo feeding experiments. Four artificial diets and five biowastes were digested using the model, and diet digestibility and supernatant nutrient contents were determined. This approach was able to distinguish broadly the worst and best performing rearing diets. However, for some of the diets, the performance estimated based on in vitro results did not match with the results of the feeding experiments. Future studies should try to establish a stronger correlation by considering fly larvae nutrient requirements, hemicellulose digestion, and the diet/gut microbiota. In vitro digestion models could be a powerful tool for academia and industry to increase conversion performance of biowastes with black soldier fly larvae.

Cell death during complete metamorphosis.

In insects that undergo complete metamorphosis, cell death is essential for reshaping or removing larval tissues and organs, thus contributing to formation of the adult's body structure. In the last few decades, the study of metamorphosis in Lepidoptera and Diptera has provided broad information on the tissue remodelling processes that occur during larva-pupa-adult transition and made it possible to unravel the underlying regulatory pathways. This review summarizes recent knowledge on cell death mechanisms in Lepidoptera and other holometabolous insects, highlighting similarities and differences with Drosophila melanogaster, and discusses the role of apoptosis and autophagy in this developmental setting. This article is part of the theme issue 'The evolution of complete metamorphosis'.

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.

The amazing complexity of insect midgut cells: types, peculiarities, and functions.

The insect midgut epithelium represents an interface between the internal and the external environment and it is the almost unique epithelial tissue by which these arthropods acquire nutrients. This epithelium is indeed able to produce digestive enzymes and to support vectorial transport of small organic nutrients, ions, and water. Moreover, it plays a key role in the defense against pathogenic microorganisms and in shaping gut microbiota. Another important midgut function is the ability to produce signaling molecules that regulate its own physiology and the activity of other organs. The two main mature cell types present in the midgut of all insects, i.e., columnar and endocrine cells, are responsible for these functions. In addition, stem cells, located at the base of the midgut epithelium, ensure the growth and renewal of the midgut during development and after injury. In insects belonging to specific orders, midgut physiology is deeply conditioned by the presence of unique cell types, i.e., goblet and copper cells, which confer peculiar features to this organ. This review reports current knowledge on the cells that form the insect midgut epithelium, focusing attention on their morphological and functional features. Notwithstanding the apparent structural simplicity of this organ, the properties of the cells make the midgut a key player in insect development and homeostasis.

A First Attempt to Produce Proteins from Insects by Means of a Circular Economy.

The worldwide growing consumption of proteins to feed humans and animals has drawn a considerable amount of attention to insect rearing. Insects reared on organic wastes and used as feed for monogastric animals can reduce the environmental impact and increase the sustainability of meat/fish production. In this study, we designed an environmentally closed loop for food supply in which fruit and vegetable waste from markets became rearing substrate for Hermetia illucens (BSF- black soldier fly). A vegetable and fruit-based substrate was compared to a standard diet for Diptera in terms of larval growth, waste reduction index, and overall substrate degradation. Morphological analysis of insect organs was carried out to obtain indications about insect health. Processing steps such as drying and oil extraction from BSF were investigated. Nutritional and microbiological analyses confirmed the good quality of insects and meal. The meal was then used to produce fish feed and its suitability to this purpose was assessed using trout. Earthworms were grown on leftovers of BSF rearing in comparison to a standard substrate. Chemical analyses of vermicompost were performed. The present research demonstrates that insects can be used to reduce organic waste, increasing at the same time the sustainability of aquaculture and creating interesting by-products through the linked bio-system establishment.

The digestive system of the adult Hermetia illucens (Diptera: Stratiomyidae): morphological features and functional properties.

The larvae of the black soldier fly (BSF), Hermetia illucens (Linnaeus, 1758) (Diptera: Stratiomyidae), are considered an efficient system for the bioconversion of organic waste into usable products, such as insect protein for animal feed and bioactive molecules. Despite the great interest toward H. illucens and its biotechnological applications, information on the biology of this insect is still scarce. In particular, no data on the structural and functional properties of the digestive system of the adult insect are available and it is a common belief that the fly does not eat. In the present work, we therefore investigate the remodeling process of the BSF larval midgut during metamorphosis, analyze the morphofunctional properties of the adult midgut, evaluate if the fly is able to ingest and digest food and assess whether the feeding supply influences the adult performances. Our results show that the larval midgut of H. illucens is removed during metamorphosis and a new pupal-adult epithelium, characterized by peculiar features compared to the larval organ, is formed by proliferation and differentiation of midgut stem cells. Moreover, our experiments indicate that the adult insect possesses a functional digestive system and that food administration affects the longevity of the fly. These data not only demonstrate that the adult BSF is able to eat but also open up the possibility to manipulate the feeding substrate of the fly to improve its performances in mass rearing procedures.