Umut Toprak

New Insights into Peritrophic Matrix Synthesis, Architecture, and Function

The peritrophic matrix (PM) is a chitin and glycoprotein layer that lines the invertebrate midgut. Although structurally different, it is functionally similar to the mucous secretions of the vertebrate digestive tract. The PM is a physical barrier, protecting the midgut epithelium from abrasive food particles, digestive enzymes, and pathogens infectious per os. It is also a biochemical barrier, sequestering and, in some cases, inactivating ingested toxins. Finally, the PM compartmentalizes digestive processes, allowing for efficient nutrient acquisition and reuse of hydrolytic enzymes. The PM consists of an organized lattice of chitin fibrils held together by chitin binding proteins. Glycans fill the interstitial spaces, creating a molecular sieve, the properties of which are dependent on the immediate ion content and pH. In this review, we have integrated recent structural and functional information to create a holistic model for the PM. We also show how this information may generate novel technologies for use in insect pest management.

A chitin deacetylase and putative insect intestinal lipases are components of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix.

One‐ and two‐dimensional gel electrophoresis coupled with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) was used to identify cDNA encoding a chitin deacetylase (McCDA1) and three insect intestinal lipases (McIIL1, McIIL2 and McIIL3) associated with the Mamestra configurata (bertha armyworm) peritrophic matrix. Recombinant McCDA1 was active and chitin deacetylase activities were detected in the midgut. McCDA1 and the McIIL genes were expressed exclusively in the midgut; however, McCDA1 and McIIL2 were expressed in all larval stages, whereas McIIL1 was expressed mainly in feeding larvae and McIIL3 primarily during the moult.

Insect intestinal mucins and serine proteases associated with the peritrophic matrix from feeding, starved and moulting Mamestra configurata larvae

Insect intestinal mucins (McIIM2‐4) expressed in the midgut of feeding, starved and moulting Mamestra configurata larvae were identified. McIIM2 and McIIM4 were associated with the peritrophic matrix (PM). PMs from feeding and starved larvae were translucent and contained organized chitin bundles perpendicular to their long axis, whereas PM from moulting larvae consisted of an inner opaque mass surrounded by an outer translucent sleeve. Serine protease genes (McSP1, McSP2, McSP25 and McSP29) were also expressed in these larvae and several serine proteases were associated with the PM. Serine protease activity was also detected in the midgut of feeding, starved and moulting larvae.

Role of enhancin in Mamestra configurata nucleopolyhedrovirus virulence: selective degradation of host peritrophic matrix proteins

To infect per os, baculovirus virions cross the peritrophic matrix (PM) to reach the midgut epithelium. Insect intestinal mucins (IIMs) are PM proteins that protect the PM and aid passage of the food bolus through the gut. Some baculoviruses, including Mamestra configurata nucleopolyhedrovirus (MacoNPV-A), encode metalloproteases, known as enhancins, that facilitate infection by degrading IIMs. We examined the interaction between MacoNPV-A enhancin and M. configurata IIMs both in vivo and in vitro. Per os inoculation of M. configurata larvae with MacoNPV-A occlusion bodies (OBs) resulted in the degradation of McIIM4 within 4 h of OB ingestion, while McIIM2 was unaffected. The PM recovered by 8 h post-inoculation. To investigate whether enhancin was responsible for the degradation of IIM, a recombinant Autographa californica multiple nucleopolyhedrovirus expressing MacoNPV enhancin (AcMNPV-enMP2) was constructed. Enhancin was found to be a component of occlusion-derived virions in AcMNPV-enMP2 and MacoNPV-A. In in vitro assays, McIIM4 was degraded after MacoNPV-A and AcMNPV-enMP2 treatments. Degradation of McIIM4 was inhibited by EDTA, a metalloprotease inhibitor, indicating that the degradation was due to enhancin activity. Thus, MacoNPV-A enhancin is able to degrade major structural PM proteins, but exhibits target substrate specificity.

Expression patterns of genes encoding proteins with peritrophin A domains and protein localization in Mamestra configurata.

Genes encoding three proteins (McPPAD1-3) with peritrophin A chitin-binding domains (PADs) were identified from a Mamestra configurata larval midgut cDNA library. In addition to midgut, McPPAD1-3 and a previously identified gene encoding the peritrophin, McPM1, were expressed in foregut, hindgut, Malpighian tubules, tracheae, fat body and cuticle; however, the corresponding McPPAD proteins exhibited different localization patterns. McPPAD1 was restricted to the digestive tract and Malpighian tubules, McPPAD2 to Malpighian tubules, and McPPAD3 to the foregut, midgut, hindgut, tracheae and cuticle. Protein fold recognition analysis using tachycitin as a guide structure modelled the McPPAD1 PADs, but not McPPAD2 or McPPAD3 PADs. The McPPAD1 PADs were predicted to contain three anti-parallel β-sheets and a hevein-like fold that form a chitin-binding pocket containing two hydrophobic R-groups in a sandwich-like orientation.

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract  The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double‐stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase‐B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase‐B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double‐stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.

Identification of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix proteins and enzymes involved in peritrophic matrix chitin metabolism.

The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America. Liquid chromatography‐tandem mass spectrometry analyses of the PM identified 82 proteins classified as: (i) peritrophins, including a new class with a CBDIII domain; (ii) enzymes involved in chitin modification (chitin deacetylases), digestion (serine proteases, aminopeptidases, carboxypeptidases, lipases and α‐amylase) or other reactions (β‐1,3‐glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase); (iii) a heterogenous group consisting of polycalin, REPATs, serpin, C‐Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut. cDNAs encoding chitin synthase‐2 (McCHS‐2), chitinase (McCHI), and β‐N‐acetylglucosaminidase (McNAG) enzymes, involved in PM chitin metabolism, were also identified. McCHS‐2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS‐2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.

Identification of a putative antifreeze protein gene that is highly expressed during preparation for winter in the sunn pest, Eurygaster maura

A cDNA library generated from the fat body of field-collected, diapausing adults of the sunn pest, Eurygaster maura revealed the presence of a transcript that encodes a protein that shares the distinct physiochemical and structural features of an insect antifreeze protein. The transcript, which is most abundant in the midgut, accumulates in adults as they leave the fields in late summer and migrate to surrounding mountainous areas to overwinter. Transcript abundance again declines when adults return to the fields the following spring. This winter pattern of abundance suggests that this protein may be critical for winter survival in the cold regions where the bug enters its obligatory diapause.

Identification and coordinated expression of perilipin genes in the biological cycle of sunn pest, Eurygaster maura (Hemiptera: Scutelleridae): Implications for lipolysis and lipogenesis

The sunn pest, Eurygaster spp., is one of the most destructive pests of grains in Asia, Europe and Africa. The nymphs and adults feed voraciously in the field by late-spring, followed by migration of adults into mountains for diapause, which includes estivation by late summer and hibernation during winter. Adults migrate back to the field by the end of diapause in mid-spring, where they mate and lay eggs. To understand how sunn pest survives and maintains basic metabolic functions without feeding for 7 months during diapause, this study focused on lipid metabolism as the major source of energy production, and the primary organ of lipid metabolism, the fat body. Studies on lipid metabolism revealed two major factors referred to perilipin protein family, Lipid Storage Droplet Protein 1 (LSD1) and Lipid Storage Droplet Protein 2 (LSD2), which are involved in hydrolysis and accumulation of lipids, respectively. In this study, two LSD (EmLSD1-2) orthologues in the hemimetabolous Eurygaster maura were identified. EmLSD1 and EmLSD2 genes were expressed in multiple tissues, but primarily in fat body. Both genes were continuously expressed throughout the insects life cycle but peaked in the 4th nymphal stage. Their expression patterns were in accordance with the biological roles of LSDs. EmLSD1 expression peaked in non-feeding stages supporting its lipolytic role, while the highest level of EmLSD2 expression was in feeding stages supporting its lipogenetic role. Expression patterns of both genes differed in females and males. Overall, expression patterns of EmLSDs provide clues to understanding the interesting life cycle of sunn pest.

Lepidopteran Peritrophic Matrix Composition, Function, and Formation

Lepidopteran larvae possess a robust digestive system featuring a multitude of hydrolytic enzymes that are able to accommodate an often highly polyphagous diet. Additional digestive complexity arises from the peritrophic matrix (PM) which encases the food bolus and compartmentalizes digestive processes. This review focuses on genomic and proteomic studies from several species that have identified what is likely to be the entire complement of proteins associated with the lepidopteran PM. In the process, a basal set of structural proteins common to the lepidopteran PM is described, and the roles of these proteins in PM structure and function are discussed. Finally, updated models for PM molecular architecture and formation which incorporate information about recently discovered proteins are provided.