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.

Yellow Mealworm Larvae (Tenebrio molitor) Fed Mycotoxin-Contaminated Wheat-A Possible Safe, Sustainable Protein Source for Animal Feed?

The aim of this study was to determine the potential for accumulation of deoxynivalenol (DON) in yellow mealworm larvae (Tenebrio molitor) reared on high DON Fusarium-infected wheat and investigate the effects on production, survival and nutritional traits. Wheat containing 200 µg/kg DON was used as the control diet. A different source of wheat was sorted into six fractions and mixed to obtain low (2000 µg/kg), medium (10,000 µg/kg) and high (12,000 µg/kg) levels of DON. Each diet was replicated five times with 300 or 200 mealworms per replicate for the feeding and breeding trials, respectively. Trial termination occurred when the first two pupae were observed (32-34 days). There was no difference in the concentrations of DON detected in the larvae between diets that ranged from 122 ± 19.3 to 136 ± 40.5 µg/kg (p = 0.88). Excretion of DON was 131, 324, 230 and 742 µg/kg for control, low, medium and high, respectively. Nutritional analysis of larvae showed maximum crude protein of 52% and crude fat of 36%. Ash, fiber, chitin, fatty-acids and amino-acid content were consistent across diets. Survival was greater than 96% for all life stages and average daily gain ranged from 1.9 ± 0.1 to 2.1 ± 0.1 mg/day per mealworm. Larvae accumulated low levels of DON from Fusarium-infected wheat diets suggesting contaminated wheat could be used to produce a sustainable, safe protein source.

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 (ß-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 ß-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.

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

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.

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 ß-sheets and a hevein-like fold that form a chitin-binding pocket containing two hydrophobic R-groups in a sandwich-like orientation.

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.

Male accessory gland secretions: modulators of female reproductive physiology and behavior.

Secretions of male accessory glands contain a variety of bioactive molecules. When transferred during mating, these molecules exert wide-ranging effects on female reproductive activity and they improve the male's chances of siring a significant proportion of the female's offspring. The accessory gland secretions may affect virtually all aspects of the female's reproductive activity. The secretions may render her unwilling or unable to remate for some time, facilitating sperm storage and ensuring that any eggs laid will be fertilized by that male's sperm. They may stimulate an increase in the number and rate of development of eggs and modulate ovulation and/or oviposition. Antimicrobial agents in the secretions ensure that the female reproductive tract is a hospitable environment during sperm transfer. In a few species the secretions include noxious chemicals. These are sequestered by developing eggs that are thereby protected from predators and pathogens when laid.

Structure of the accessory reproductive glands of the male migratory grasshopper, Melanoplus sanguinipes.

The accessory reproductive glands of Melanoplus sanguinipes comprise two bilateral masses of 16 tubules each, distinguishable in sexually mature insects as four white, ten short hyaline, one long hyaline, and a seminal vesicle. Over most of its length, the wall of each tubule consists of a simple glandular epithelium resting on a basal lamina, surrounded by a thin layer of circular muscle. However, near the junction with the ejaculatory duct, the wall of each tubule has a much thickened circular muscle layer and squamous or cuboidal epithelium, the region serving to regulate movement of secretion into the ejaculatory duct. Interdigitation of adjacent epithelial cells is common, and several kinds of specialized junctions occur. In the glandular region, all epithelial cells appear the same and may be flattened, cuboidal, or columnar depending on the tubule type. Except for those of the seminal vesicle, the glandular epithelial cells share ultrastructural features typical of cells engaged in the synthesis of protein for export. Despite these general similarities, in most instances subtle differences occur in the cellular ultrastructure of the epithelia of each tubule and in the appearance of their luminal secretions, suggesting that the tubules are functionally specialized.