MosChito rafts as a promising biocontrol tool against larvae of the common house mosquito, Culex pipiens.

Mosquito control is of paramount importance, in particular, in light of the major environmental alterations associated with human activities, from climate change to the altered distribution of pathogens, including those transmitted by Arthropods. Here, we used the common house mosquito, Culex pipiens to test the efficacy of MosChito raft, a novel tool for mosquito larval control. MosChito raft is a floating hydrogel matrix, composed of chitosan, genipin and yeast cells, as bio-attractants, developed for the delivery of a Bacillus thuringiensis israeliensis (Bti)-based bioinsecticide to mosquito larvae. To this aim, larvae of Cx. pipiens were collected in field in Northern Italy and a novel colony of mosquito species (hereafter: Trescore strain) was established. MosChito rafts, containing the Bti-based formulation, were tested on Cx. pipiens larvae from the Trescore strain to determine the doses to be used in successive experiments. Thus, bioassays with MosChito rafts were carried out under semi-field conditions, both on larvae from the Trescore strain and on pools of larvae collected from the field, at different developmental stages. Our results showed that MosChito raft is effective against Cx. pipiens. In particular, the observed mortality was over 50% after two days exposure of the larvae to MosChito rafts, and over 70-80% at days three to four, in both laboratory and wild larvae. In conclusion, our results point to the MosChito raft as a promising tool for the eco-friendly control of a mosquito species that is not only a nuisance insect but is also an important vector of diseases affecting humans and animals.

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.

MosChito rafts as effective and eco-friendly tool for the delivery of a Bacillus thuringiensis-based insecticide to Aedes albopictus larvae.

Adult mosquito females, through their bites, are responsible for the transmission of different zoonotic pathogens. Although adult control represents a pillar for the prevention of disease spread, larval control is also crucial. Herein we characterized the effectiveness of a suitable tool, named "MosChito raft", for the aquatic delivery of a Bacillus thuringiensis var. israelensis (Bti) formulate, a bioinsecticide active by ingestion against mosquito larvae. MosChito raft is a floating tool composed by chitosan cross-linked with genipin in which a Bti-based formulate and an attractant have been included. MosChito rafts (i) resulted attractive for the larvae of the Asian tiger mosquito Aedes albopictus, (ii) induced larval mortality within a few hours of exposure and, more importantly, (iii) protected the Bti-based formulate, whose insecticidal activity was maintained for more than one month in comparison to the few days residual activity of the commercial product. The delivery method was effective in both laboratory and semi-field conditions, demonstrating that MosChito rafts may represent an original, eco-based and user-friendly solution for larval control in domestic and peri-domestic aquatic habitats such as saucers and artificial containers in residential or urban environments.

Cuticle Modifications and Over-Expression of the Chitin-Synthase Gene in Diflubenzuron-Resistant Phenotype.

Insecticide resistance is a major threat challenging the control of harmful insect species. The study of resistant phenotypes is, therefore, pivotal to understand molecular mechanisms underpinning insecticide resistance and plan effective control and resistance management strategies. Here, we further analysed the diflubenzuron (DFB)-resistant phenotype due to the point-mutation I1043M in the chitin-synthase 1 gene (chs1) in the mosquito Culex pipiens. By comparing susceptible and resistant strains of Cx. pipiens through DFB bioassays, molecular analyses and scanning electron microscopy, we showed that the I1043M-resistant mosquitoes have: (i) a striking level of DFB resistance (i.e., resistance ratio: 9006); (ii) a constitutive 11-fold over-expression of the chs1 gene; (iii) enhanced cuticle thickness and cuticular chitin content. Culex pipiens is one of the most important vector species in Europe and the rapid spread of DFB resistance can threaten its control. Our results, by adding new data about the DFB-resistant phenotype, provide important information for the control and management of insecticide resistance.

Biodegradable floating hydrogel baits as larvicide delivery systems against mosquitoes.

Biological methods for mosquito larvae control are completely biodegradable and have null or limited effects on nontarget organisms. However, commercially available products have a low residual activity, with the consequent need for multiple applications that inevitably increase costs and the risk of resistance phenomena insurgence. Smart delivery systems made of hydrogels proved their efficacy in increasing the action duration of biolarvicides up to several months, but the lack of an efficient baiting mechanism to strongly attract the target pest remains a problem in practical applications. In this work, we investigated two novel hydrogel-based formulations of completely natural composition for baiting and killing larvae of Aedes albopictus mosquitos. The proposed materials consist of charged crosslinked polysaccharides (chitosan and cellulose) and are specifically manufactured to float in water, simulating organic matter usually present at breeding sites. Within the hydrogels' matrix, yeast colonies of Saccharomyces cerevisiae were embedded as phagostimulants alongside a biolarvicide (Bacillus thuringiensis var. israelensis (Bti)). Despite the similar chemical nature and structure, chitosan-based hydrogels exhibited a markedly superior baiting potential compared to those made of cellulose and also succeeded in efficiently killing mosquito larvae just after a few hours from administration. We are confident that the proposed smart delivery hydrogel made of chitosan can be an enabling tool to attract mosquito larvae towards biopesticides of different nature without delocalizing active ingredients away from the breeding site and to simultaneously increase their residual activity, thus holding the potential of minimizing environmental pollution related to pest control and vector-borne disease prevention.

Mosquito Trilogy: Microbiota, Immunity and Pathogens, and Their Implications for the Control of Disease Transmission.

In mosquitoes, the interaction between the gut microbiota, the immune system, and the pathogens that these insects transmit to humans and animals is regarded as a key component toward the development of control strategies, aimed at reducing the burden of severe diseases, such as malaria and dengue fever. Indeed, different microorganisms from the mosquito microbiota have been investigated for their ability to affect important traits of the biology of the host insect, related with its survival, development and reproduction. Furthermore, some microorganisms have been shown to modulate the immune response of mosquito females, significantly shaping their vector competence. Here, we will review current knowledge in this field, focusing on i) the complex interaction between the intestinal microbiota and mosquito females defenses, both in the gut and at humoral level; ii) how knowledge on these issues contributes to the development of novel and targeted strategies for the control of mosquito-borne diseases such as the use of paratransgenesis or taking advantage of the relationship between Wolbachia and mosquito hosts. We conclude by providing a brief overview of available knowledge on microbiota-immune system interplay in major insect vectors.

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.

Venomics of the ectoparasitoid wasp Bracon nigricans.

BACKGROUND: Venom is one of the most important sources of regulation factors used by parasitic Hymenoptera to redirect host physiology in favour of the developing offspring. This has stimulated a number of studies, both at functional and "omics" level, which, however, are still quite limited for ectophagous parasitoids that permanently paralyze and suppress their victims (i.e., idiobiont parasitoids). RESULTS: Here we present a combined transcriptomic and proteomic study of the venom of the generalist idiobiont wasp Bracon nigricans, an ectophagous larval parasitoid of different lepidopteran species, for which we recently described the host regulation strategy and the functional role of the venom in the induction of physiological changes in parasitized hosts. The experimental approach used led to the identification of the main components of B. nigricans venom involved in host regulation. Enzymes degrading lipids, proteins and carbohydrates are likely involved in the mobilization of storage nutrients from the fat body and may concurrently be responsible for the release of neurotoxic fatty acids inducing paralysis, and for the modulation of host immune responses. CONCLUSION: The present work contributes to fill the gap of knowledge on venom composition in ectoparasitoid wasps, and, along with our previous physiological study on this species, provides the foundation on which to develop a functional model of host regulation, based both on physiological and molecular data. This paves the way towards a better understanding of parasitism evolution in the basal lineages of Hymenoptera and to the possible exploitation of venom as source of bioinsecticidal molecules.

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.

Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp.

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably influenced by the acquisition of a large number of genes by horizontal gene transfer (HGT) across different kingdoms. However, in depth-studies on the physiological traits conferred by these accidental DNA acquisitions are largely lacking. Here we elucidate the functional role of Sl gasmin, a gene of a symbiotic virus of a parasitic wasp that has been transferred to an ancestor of the moth species Spodoptera littoralis and domesticated. This gene is highly expressed in circulating immune cells (haemocytes) of larval stages, where its transcription is rapidly boosted by injection of microorganisms into the body cavity. RNAi silencing of Sl gasmin generates a phenotype characterized by a precocious suppression of phagocytic activity by haemocytes, which is rescued when these immune cells are incubated in plasma samples of control larvae, containing high levels of the encoded protein. Proteomic analysis demonstrates that the protein Sl gasmin is released by haemocytes into the haemolymph, where it opsonizes the invading bacteria to promote their phagocytosis, both in vitro and in vivo. Our results show that important physiological traits do not necessarily originate from evolution of pre-existing genes, but can be acquired by HGT events, through unique pathways of symbiotic evolution. These findings indicate that insects can paradoxically acquire selective advantages with the help of their natural enemies.

Structural and Functional Characterization of Hermetia illucens Larval Midgut.

The larvae of Hermetia illucens are among the most promising agents for the bioconversion of low-quality biomass, such as organic waste, into sustainable and nutritionally valuable proteins for the production of animal feed. Despite the great interest in this insect, the current literature provides information limited to the optimization of rearing methods for H. illucens larvae, with particular focus on their efficiency in transforming different types of waste and their nutritional composition in terms of suitability for feed production. Surprisingly, H. illucens biology has been neglected and a deep understanding of the morphofunctional properties of the larval midgut, the key organ that determines the extraordinary dietary plasticity of this insect, has been completely overlooked. The present study aims to fill this gap of knowledge. Our results demonstrate that the larval midgut is composed of distinct anatomical regions with different luminal pH and specific morphofunctional features. The midgut epithelium is formed by different cell types that are involved in nutrient digestion and absorption, acidification of the lumen of the middle region, endocrine regulation, and growth of the epithelium. A detailed characterization of the activity of enzymes involved in nutrient digestion and their mRNA expression levels reveals that protein, carbohydrate, and lipid digestion is associated to specific regions of this organ. Moreover, a significant lysozyme activity in the lumen of the anterior and middle regions of the midgut was detected. This enzyme, together with the strong acidic luminal pH of middle tract, may play an important role in killing pathogenic microorganisms ingested with the feeding substrate. The evidence collected led us to propose a detailed functional model of the larval midgut of H. illucens in which each region is characterized by peculiar features to accomplish specific functions. This platform of knowledge sets the stage for developing rearing protocols to optimize the bioconversion ability of this insect and its biotechnological applications.

The Intestinal Microbiota of Hermetia illucens Larvae Is Affected by Diet and Shows a Diverse Composition in the Different Midgut Regions.

The larva of the black soldier fly (Hermetia illucens) has emerged as an efficient system for the bioconversion of organic waste. Although many research efforts are devoted to the optimization of rearing conditions to increase the yield of the bioconversion process, microbiological aspects related to this insect are still neglected. Here, we describe the microbiota of the midgut of H. illucens larvae, showing the effect of different diets and midgut regions in shaping microbial load and diversity. The bacterial communities residing in the three parts of the midgut, characterized by remarkable changes in luminal pH values, differed in terms of bacterial numbers and microbiota composition. The microbiota of the anterior part of the midgut showed the highest diversity, which gradually decreased along the midgut, whereas bacterial load had an opposite trend, being maximal in the posterior region. The results also showed that the influence of the microbial content of ingested food was limited to the anterior part of the midgut, and that the feeding activity of H. illucens larvae did not significantly affect the microbiota of the substrate. Moreover, a high protein content compared to other macronutrients in the feeding substrate seemed to favor midgut dysbiosis. The overall data indicate the importance of taking into account the presence of different midgut structural and functional domains, as well as the substrate microbiota, in any further study that aims at clarifying microbiological aspects concerning H. illucens larval midgut.IMPORTANCE The demand for food of animal origin is expected to increase by 2050. Since traditional protein sources for monogastric diets are failing to meet the increasing demand for additional feed production, there is an urgent need to find alternative protein sources. The larvae of Hermetia illucens emerge as efficient converters of low-quality biomass into nutritionally valuable proteins. Many studies have been performed to optimize H. illucens mass rearing on a number of organic substrates and to quantitatively and qualitatively maximize the biomass yield. On the contrary, although the insect microbiota can be fundamental for bioconversion processes and its characterization is mandatory also for safety aspects, this topic is largely overlooked. Here, we provide an in-depth study of the microbiota of H. illucens larval midgut, taking into account pivotal aspects, such as the midgut spatial and functional regionalization, as well as microbiota and nutrient composition of the feeding substrate.

Host regulation by the ectophagous parasitoid wasp Bracon nigricans.

The host regulation process has been widely investigated in endophagous parasitoid wasps, which in most cases finely interact with living hosts (i.e. koinobiont parasitoids). In contrast, only very limited information is available for ectophagous parasitoids that permanently paralyze and rapidly suppress their victims (i.e. idiobiont parasitoids). Here we try to fill this research gap by investigating the host regulation by Bracon nigricans, an ectophagous idiobiont wasp species. Parasitism, mainly by venom action, is able to redirect host metabolism in order to enhance its nutritional suitability for the developing parasitoid larvae and to provide the required metabolic support to host tissues. The observed alterations of the host titers of haemolymph proteins, carbohydrates and acylglycerols are associated with a parasitoid-induced mobilization of nutrients stored in the fat body. This tissue undergoes a controlled degradation mediated by a close surface interaction with haemocytes, where a cathepsin L activity is localized, as demonstrated by immunolocalization, biochemical and transcriptional data. B. nigricans parasitism does not markedly influence the survival of haemocytes, even though a persistent suppression of the immune competence is observed in parasitized hosts, which show a reduced capacity to encapsulate and melanize non-self objects. These immune alterations likely allow a more efficient food uptake and use by the ectophagous larvae. The obtained results indicate that the host regulation process in basal lineages of parasitic Hymenoptera is more complex than expected and shares functional similarities with adaptive strategies occurring in derived koinobiont species.

Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism.

Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host.

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.

Effects of Trichoderma viride chitinases on the peritrophic matrix of Lepidoptera.

BACKGROUND: The peritrophic matrix (PM) is formed by a network of chitin fibrils associated with proteins, glycoproteins and proteoglycans that lines the insect midgut. It is a physical barrier involved in digestion processes, and protects the midgut epithelium from food abrasion, pathogen infections and toxic materials. Given its fundamental role in insect physiology, the PM represents an excellent target for pest control strategies. Although a number of viral, bacterial and insect chitinolytic enzymes affecting PM integrity have already been tested, exploitation of fungal chitinases has been almost neglected. Fungal chitinases, already in use as fungal phytopathogen biocontrol agents, are known to attack the insect cuticle, but their action on the insect gut needs to be better investigated. RESULTS: In the present paper, we performed a biochemical characterisation of a commercial mixture of chitinolytic enzymes derived from Trichoderma viride and analysed its in vitro and in vivo effects on the PM of the silkworm Bombyx mori, a model system among Lepidoptera. We found that these enzymes have significant in vitro effects on the structure and permeability of the PM of this insect. A bioassay supported these results and showed that the oral administration of the mixture causes PM alterations, leading to adverse consequences on larval growth and development, negatively affecting pupal weight and even inducing mortality. CONCLUSIONS: This study provides an integrated experimental approach to evaluate the effects of fungal chitinases on Lepidoptera. The encouraging results obtained herein make us confident about the possible use of fungal chitinases to control lepidopteran pests.

The midgut of the silkmoth Bombyx mori is able to recycle molecules derived from degeneration of the larval midgut epithelium.

The midgut represents the middle part of the alimentary canal and is responsible for nutrient digestion and absorption in insect larva. Despite the growing interest in this organ for different purposes, such as studies on morphogenesis and differentiation, stem cell biology, cell death processes and transport mechanisms, basic information on midgut development is still lacking for a large proportion of insect species. Undoubtedly, this lack of data could hinder the full exploitation of practical applications that involve midgut as their primary target. This may represent in particular a significant problem for Lepidoptera, an insect order that includes some of the most important species of high economic importance. With the aim of overcoming this fragmentation of knowledge, we performed a detailed morphofunctional analysis of the midgut of the silkworm, Bombyx mori, a representative model among Lepidoptera, during its development from the larval up to the adult stage, focusing attention on stem cells. Our data demonstrate stem cell proliferation and differentiation, not only in the larval midgut but also in the pupal and adult midgut epithelium. Moreover, we present evidence for a complex trophic relationship between the dying larval epithelium and the new adult one, which is established during metamorphosis. This study, besides representing the first morphological and functional characterization of the changes that occur in the midgut of a lepidopteron during the transition from the larva to the moth, provides a detailed analysis of the midgut of the adult insect, a stage that has been neglected up to now.

New synthesis and biological evaluation of uniflorine A derivatives: towards specific insect trehalase inhibitors.

7-Deoxy-uniflorine A (6), synthesized ex novo with a straightforward and simple strategy, and the analogues 4, 5 and 7, were evaluated as potential inhibitors of insect trehalase from Chironomus riparius and Spodoptera littoralis. All the compounds were tested against porcine trehalase as the mammalian counterpart and α-amylase from human saliva as a relevant glucolytic enzyme. The aim of this work is the identification of the simplest pyrrolizidine structure necessary to impart selective insect trehalase inhibition, in order to identify new specific inhibitors that can be easily synthesized compared to our previous reports with the potential to act as non-toxic insecticides and/or fungicides. All the derivatives 4­7 proved to be active (from low micromolar to high nanomolar range activity) towards insect trehalases, while no activity was observed against α-amylase. In particular, the natural compound uniflorine A and its 7-deoxy analogue were found to selectively inhibit insect trehalases, as they are inactive towards the mammalian enzyme. The effect of compound 6 was also analyzed in preliminary in vivo experiments. These new findings allow the identification of natural uniflorine A and its 7-deoxy analogue as the most promising inhibitors among a series of pyrrolizidine derivatives for future development in the agrochemical field, and the investigation also outlined the importance of the stereochemistry at C-6 of pyrrolizidine nucleus to confer such enzyme specificity.

A virulence factor encoded by a polydnavirus confers tolerance to transgenic tobacco plants against lepidopteran larvae, by impairing nutrient absorption.

The biological control of insect pests is based on the use of natural enemies. However, the growing information on the molecular mechanisms underpinning the interactions between insects and their natural antagonists can be exploited to develop "bio-inspired" pest control strategies, mimicking suppression mechanisms shaped by long co-evolutionary processes. Here we focus on a virulence factor encoded by the polydnavirus associated with the braconid wasp Toxoneuron nigriceps (TnBV), an endophagous parasitoid of noctuid moth larvae. This virulence factor (TnBVANK1) is a member of the viral ankyrin (ANK) protein family, and appears to be involved both in immunosuppression and endocrine alterations of the host. Transgenic tobacco plants expressing TnBVANK1 showed insecticide activity and caused developmental delay in Spodoptera littoralis larvae feeding on them. This effect was more evident in a transgenic line showing a higher number of transcripts of the viral gene. However, this effect was not associated with evidence of translocation into the haemocoel of the entire protein, where the receptors of TnBVANK1 are putatively located. Indeed, immunolocalization experiments evidenced the accumulation of this viral protein in the midgut, where it formed a thick layer coating the brush border of epithelial cells. In vitro transport experiments demonstrated that the presence of recombinant TnBVANK1 exerted a dose-dependent negative impact on amino acid transport. These results open new perspectives for insect control and stimulate additional research efforts to pursue the development of novel bioinsecticides, encoded by parasitoid-derived genes. However, future work will have to carefully evaluate any effect that these molecules may have on beneficial insects and on non-target organisms.