Eleonora Franzetti

Molecular cloning, characterization and expression analysis of ATG1 in the silkworm, Bombyx mori

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process. In the present study we isolated two full-length cDNAs of 2175 (transcript variant A) and 2271 (transcript variant B) bases representing ATG1 in the silkworm. Phylogenetic analysis indicated that BmATG1 was closely related to orthologs of other insects. The encoded BmAtg1 proteins shared extensive homology with orthologs from yeast to mammals, showing high conservation at the N-terminal region where the catalytic domain and ATP- and Mg-binding sites are located. A de novo prediction of the three-dimensional structure for each protein is presented. We used real-time RT-PCR to quantify dynamic changes in mRNA copy number of BmATG1 in the midgut and fat body of fifth instar larvae undergoing starvation, as well as in other tissues of silkworm at the end of last larval instar. Our qPCR results revealed that BmATG1 expression levels at the end of larval life were comparable in the midgut, fat body and Malpighian tubules, while these were higher in the gonads; moreover, the mRNA copy number of ATG1 was very different among the anterior, middle and posterior silk glands. Real-time PCR analysis also showed that starvation significantly influenced BmATG1 mRNA copy number in the fat body of silkworm, inducing an upregulation 24h after food withdrawal, with only a slight effect in the midgut. Low expression levels of BmATG1 were observed in both tissues of control animals up to the second day of spinning phase.

Identification of Enterococcus mundtii as a pathogenic agent involved in the “flacherie” disease in Bombyx mori L. larvae reared on artificial diet

Enterococcus mundtii was shown to be directly correlated with flacherie disease of the silkworm larvae reared on artificial diet supplemented with chloramphenicol. Its identification was carried out by means of light and electron microscopy and nucleotide sequencing of 16S gene. The bacterium is capable of rapidly multiplying in the silkworm gut and of invading other body tissues, as demonstrated by deliberate infection of germfree larvae and by subsequent TEM observations. E. mundtii can endure alkaline pH of the silkworm gut and it has been proved to adapt in vitro to commonly applied doses of chloramphenicol, whose use can further contribute to reduce competition by other bacteria in Bombyx mori alimentary canal. The modality of transmission of the infection to the larvae was among the objectives of the present research. Since contamination of the progeny by mother moths can be avoided through routine egg shell disinfection, a trans-ovarian vertical transmission can be ruled out. On the other hand the bacterium was for the first time identified on mulberry leaves, and therefore artificial diet based on leaf powder could be a source of infection. We showed that while microwaved diet could contain live E. mundtii cells, the autoclaved diet is safe in this respect. Being E. mundtii also part of the human-associated microbiota, and since B. mori is totally domestic species, a possible role of man in its epidemiology can be postulated.

The role of autophagy in the midgut epithelium of Eubranchipus grubii (Crustacea, Branchiopoda, Anostraca)

Eubranchipus grubii (Crustacea, Branchiopoda, Anostraca) is an omnivorous filter feeder whose life span lasts no more than 12 weeks. Adult males and females of E. grubii were used for ultrastructural studies of the midgut epithelium and an analysis of autophagy. The midgut epithelium is formed by columnar digestive cells and no regenerative cells were observed. A distinct regionalization in the distribution of organelles appears - basal, perinuclear and apical regions were distinguished. No differences in the ultrastructure of digestive cells were observed between males and females. Autophagic disintegration of organelles occurs throughout the midgut epithelium. Degenerated organelles accumulate in the neighborhood of Golgi complexes, and these complexes presumably take part in phagophore and autophagosome formation. In some cases, the phagophore also surrounds small autophagosomes, which had appeared earlier. Fusion of autophagosomes and lysosomes was not observed, but lysosomes are enclosed during autophagosome formation. Autophagosomes and autolysosomes are discharged into the midgut lumen due to apocrine secretion. Autophagy plays a role in cell survival by protecting the cell from cell death.

Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism

Significance Bacillus thuringiensis and its toxins are widely used for insect control. Notwithstanding the remarkable importance of this insect pathogen, its killing mechanism has yet to be fully elucidated. Here we show that the microbiota resident in the host midgut triggers a lethal septicemia. The infection process is enhanced by reducing the host immune response and its control on replication of midgut bacteria invading the body cavity through toxin-induced epithelial lesions. The experimental approach used, leaving the midgut microbiota unaltered, allows identification of the bacterial species switching from resident symbionts to pathogens and sets the stage for developing new insect biocontrol technologies based on host immunosuppression as a strategy to enhance the impact of natural antagonists. 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.

A Molecular View of Autophagy in Lepidoptera

Metamorphosis represents a critical phase in the development of holometabolous insects, during which the larval body is completely reorganized: in fact, most of the larval organs undergo remodeling or completely degenerate before the final structure of the adult insect is rebuilt. In the past, increasing evidence emerged concerning the intervention of autophagy and apoptosis in the cell death processes that occur in larval organs of Lepidoptera during metamorphosis, but a molecular characterization of these pathways was undertaken only in recent years. In addition to developmentally programmed autophagy, there is growing interest in starvation-induced autophagy. Therefore we are now entering a new era of research on autophagy that foreshadows clarification of the role and regulatory mechanisms underlying this self-digesting process in Lepidoptera. Given that some of the most important lepidopteran species of high economic importance, such as the silkworm, Bombyx mori, belong to this insect order, we expect that this information on autophagy will be fully exploited not only in basic research but also for practical applications.

A model of osteoblast–osteocyte kinetics in the development of secondary osteons in rabbits

The kinetics of osteogenic cells within secondary osteons have been examined within a 2‐D model. The linear osteoblast density of the osteons and the osteocyte lacunae density were compared with other endosteal lamellar systems of different geometries. The cell density was significantly greater in the endosteal appositional zone and was always flatter than the central osteonal canals. Fully structured osteons compared with early structuring (cutting cones) did not show any significant differences in density. The osteoblast density may remain constant because some of them leave the row and become embedded within matrix. The overall shape of the Haversian system represented a geometrical restraint and it was thought to be related to osteoblast–osteocyte transformation. To test this hypothesis of an early differentiation and recruitment of the osteoblast pool which completes the lamellar structure of the osteon, the number and density of osteoblasts and osteocyte lacunae were evaluated. In the central canal area, the mean osteoblast linear density and the osteocyte lacunae planar density were not significantly different among sub‐classes (with the exclusion of the osteocyte lacunae of the 300–1000 μm2 sub‐class). The mean number of osteoblasts compared with osteocyte lacunae resulted in significantly higher numbers in the two sub‐classes, no significant difference was seen in the two middle sub‐classes with the larger canals, and there were significantly lower levels in the smallest central canal sub‐class. The TUNEL technique was used to identify the morphological features of apoptosis within osteoblasts. It was found that apoptosis occurred during the late phase of osteon formation but not in osteocytes. This suggests a regulatory role of apoptosis in balancing the osteoblast–osteocyte equilibrium within secondary osteon development. The position of the osteocytic lacunae did not correlate with the lamellar pattern and the lacunae density in osteonal radial sectors was not significantly different. These findings support the hypothesis of an early differentiation of the osteoblast pool and the independence of the fibrillar lamellation from osteoblast–osteocyte transformation.

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.

Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus

The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.

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 key role of autophagy and its relationship with apoptosis in lepidopteran larval midgut remodeling

Cell death is a primary cellular response essential for the development, differentiation, homeostasis, and survival of organisms. This highly heterogeneous process, which includes apoptotic and autophagy-based cell death, can be activated by distinct biochemical cascades, and can display different morphological features. For this reason, a precise characterization of the numerous cell death modalities described so far in eukaryotes, and of their relationships, constitutes a major challenge for current research. Cell death-associated phenomena occur extensively in the larvae of holometabolous insects (i.e. Lepidoptera) during development and metamorphosis to eliminate tissues and organs that the adult does not need. Several larval organs of lepidopteran larvae have been used over the years to study autophagy and apoptosis; however, the current literature is basically fragmentary and confusing. The completion of genome sequencing in Bombyx mori and the development of molecular tools to manipulate the expression of autophagic and apoptotic genes that have now been identified in the silkworm opened up new perspectives and made it possible to analyze in-depth the cell death processes that occur in these insects. This chapter summarizes current knowledge about autophagy research in Lepidoptera. The use of the larval midgut is discussed as a model for studying the roles and regulation of autophagy, and for gaining insight as to how autophagy and apoptosis cooperate in cell death events in lepidopteran larval organs.