Fall armyworm larvae (FAW), which are serious pests in the southern United States, show retarded growth when they feed on insect-resistant maize inbreds Mp704 and Mp708. These maize genotypes are not only resistant to FAW, but to a number of other lepidopteran pests. In these genotypes, a unique, extracellular, 33-KDa cysteine protease (Mir1-CP) rapidly accumulates in the whorl in response to insect feeding. Initial morphological studies on larvae feeding on resistant maize plants over-expressing the cysteine protease showed severe damage in insect?s first line of defense, the peritrophic matrix (PM). But it is not known whether the cysteine protease has unprecedented effect on insect defense mechanisms. This study focuses on understanding the functional involvement of the cysteine protease (Mir1-CP) in a plant-herbivore defense mechanism. I used purified, recombinant 33-KDa cysteine protease (Mir1-CP) and its two mutated forms (Mut1 and Mut2) to determine their effects on the permeability of PMs from fall armyworm and other lepidopteran larvae. The purified Mir1-CP was also used to determine its minimal effective dosage on lepidopteran larval growth as well as to qualitatively determine their direct morphological effects on PM and gut regions of fall armyworm larvae. In vitro permeability studies demonstrated that the recombinant Mir1-CP directly permeabilized the PM and requires both cysteine at the active site and the terminal 25 amino acids to achieve complete permeabilization. Dose response study suggested that physiologically relevant concentrations of Mir1-CP in the maize whorl would be effective in controlling a broad range of lepidopteran pests. The study also suggested that stacking Mir1-CP and Bt-toxin (Bt-CryIIA) genes in transgenic plants could broaden the normal range of both Mir1-CP and Bt-toxin. Morphological studies using three different microscopic techniques showed damaged PM in larvae fed on Mir1-CP diet. These results suggest that by directly permeabilizing and damaging the PM, the Mir1-CP provides critical defense in host plants against lepidopteran pests.

<p>The peritrophic matrix (PM) lines the insect midgut and is composed of chitin and protein. It is required for organization of digestion and for protection of epithelial cells from mechanical damage, pathogens, and toxins. The PM of <i>Mamestra configurata</i> (Lepidoptera: Noctuidae), bertha armyworm, a serious pest of cruciferous oilseed rape, was studied. The multilayered PM is delaminated from the anterior midgut epithelium during molting Phase II by periodic pulses and degraded during the molting Phase I stage. These events are controlled by chitin synthase-B, and chitinolytic enzymes, such as chitinase and β-<i>N</i>-acetylglucosaminidase. Eighty-two PM proteins were identified and classified as: i) peritrophins, ii) enzymes and iii) other proteins. Peritrophins were further classified as simple, binary, complex and repetitive according to their structural organization and phylogenetic analysis of peritrophin A domains. The expression of most genes encoding PM proteins was specific to the midgut and independent of larval feeding status, developmental stage, or PM formation.</p> <p>This study includes the first report of chitin deacetylase (CDA) activity in the insect midgut suggesting that the PM may contain chitosan. Digestive enzymes, such as insect intestinal lipases (IILs) and serine proteases were also associated with the PM. The IIL genes differed in their expression during larval development; however, serine protease genes were expressed continuously and serine protease activity was present in the midgut of feeding and nonfeeding stages. <i>M. configurata</i> IIM4, a complex peritrophin, was susceptible to degradation by Mamestra configurata nucleopolyhedrovirus-A challenge, as the first evidence of IIM degradation by an alphabaculovirus enhancin. <i>M. configurata</i> IIM2, a binary peritrophin, was unaffected by baculoviral challenge and such resistance of an IIM has not been reported previously. The current study is also the first demonstration of silencing by RNA interference (RNAi) of any gene encoding a PM protein, in this case <i>M. configurata</i> CDA1 (McCDA1) and McPM1. In addition, both <i>in vitro</i> and <i>per os</i> feeding experiments revealed <i>McCDA1</i> silencing starting at 24 or 36 hours posttreatment, as one of the most successful demonstrations of RNAi in a lepidopteran.</p>

<p>The peritrophic matrix (PM) lines the insect midgut and is composed of chitin and protein. It is required for organization of digestion and for protection of epithelial cells from mechanical damage, pathogens, and toxins. The PM of <i>Mamestra configurata</i> (Lepidoptera: Noctuidae), bertha armyworm, a serious pest of cruciferous oilseed rape, was studied. The multilayered PM is delaminated from the anterior midgut epithelium during molting Phase II by periodic pulses and degraded during the molting Phase I stage. These events are controlled by chitin synthase-B, and chitinolytic enzymes, such as chitinase and β-<i>N</i>-acetylglucosaminidase. Eighty-two PM proteins were identified and classified as: i) peritrophins, ii) enzymes and iii) other proteins. Peritrophins were further classified as simple, binary, complex and repetitive according to their structural organization and phylogenetic analysis of peritrophin A domains. The expression of most genes encoding PM proteins was specific to the midgut and independent of larval feeding status, developmental stage, or PM formation.</p> <p>This study includes the first report of chitin deacetylase (CDA) activity in the insect midgut suggesting that the PM may contain chitosan. Digestive enzymes, such as insect intestinal lipases (IILs) and serine proteases were also associated with the PM. The IIL genes differed in their expression during larval development; however, serine protease genes were expressed continuously and serine protease activity was present in the midgut of feeding and nonfeeding stages. <i>M. configurata</i> IIM4, a complex peritrophin, was susceptible to degradation by Mamestra configurata nucleopolyhedrovirus-A challenge, as the first evidence of IIM degradation by an alphabaculovirus enhancin. <i>M. configurata</i> IIM2, a binary peritrophin, was unaffected by baculoviral challenge and such resistance of an IIM has not been reported previously. The current study is also the first demonstration of silencing by RNA interference (RNAi) of any gene encoding a PM protein, in this case <i>M. configurata</i> CDA1 (McCDA1) and McPM1. In addition, both <i>in vitro</i> and <i>per os</i> feeding experiments revealed <i>McCDA1</i> silencing starting at 24 or 36 hours posttreatment, as one of the most successful demonstrations of RNAi in a lepidopteran.</p>

Doctor of Philosophy / Department of Entomology / Marcelo Ramalho-Ortigao / The emergence and reemergence of vector-borne diseases pose significant threats to humans and other animals worldwide. Although vector control relies mostly on insecticides, the emergence of insecticide resistance urges for the development of new strategies to control the spread of such diseases. For sand fly-transmitted leishmaniasis, Transmission Blocking Vaccines (TBV) may constitute a feasible strategy to impair Leishmania transmission from infected to uninfected vertebrate hosts. Moreover, sand fly saliva-based vaccines represent an alternative or complementary approach as these vaccines protect different mammalian hosts against Leishmania. Based on the potential use of sand fly molecules as vaccines against leishmaniasis, we assessed the potential of Phlebotomus papatasi midgut secreted proteins as TBV candidates and the expression variability of sand fly salivary gland genes. Regarding the TBV approach, we took advantage of the RNA interference (RNAi) technique to evaluate the effects of knocking down P. papatasi midgut-specific genes on Leishmania major development within the sand fly midgut. Whereas peritrophin 1 (PpPer1) knock down led to increased Le. major load by 39%, knocking down chitinase 1 (PpChit1) reduced Le. major load in P. papatasi midguts by 63%. Thus, our data strongly suggest that PpChit1 constitutes a potential target for TBV approaches against Leishmania transmission in endemic areas. Concerning protective vaccines based on salivary gland secreted proteins, we searched for expression polymorphism in selected salivary gland genes in natural and colonized populations of P. papatasi. Significant differences in salivary gland gene expression were not only exhibited in P. papatasi specimens collected in different geographic habitats but also seasonal difference in gene expression was displayed by specimens belonging to the same population. As antigen dose is an important component of immune responses, different doses of salivary protein inoculated into host skin may interfere with vaccine protection. Thus, the efficacy of sand fly saliva-based vaccine upon exposure to different salivary protein doses must be evaluated before deployment in endemic areas. Our data also ruled out some biotic factors as responsible for fine-tuning the expression of such genes. Overall, this dissertation makes significant contribution to the development of sand fly-based vaccines against leishmaniasis.

anglický Phlebotomine sand flies (Diptera: Psychodidae) are the only proven vectors of Leishmania parasites (Kinetoplastida: Trypanosomatidae). In Nematoceran Diptera, including sand flies, adults produce a type 1 peritrophic matrix (PM) which is secreted in response to the distension of the midgut caused by blood meal. The PM is an acellular envelope composed of chitin fibres and proteins, which protects the midgut epithelium against abrasion and pathogens and improves digestion. In hematophagous insects, the PM also plays a central role in heme detoxification. Female sand flies acquire Leishmania with a bloodmeal and the parasites undergo complicated development in their gut finished by the colonization of the stomodeal valve. The PM is one of the most important barriers in Leishmania development and its role in the vector competence of the S. schwetzi is the main topic of this master thesis. The PM's kinetic and morfology in the S. schwetzi is compared with other three sand fly species which differ in susceptibility to L. donovani. The key role of the PM in S. schwetzi vector competence is finally proved by disrupting the PM using the exogenous chitinase from Beauveria bassiana. Under these artificial conditions, the Leishmania parasites (L. donovani and L. major) are able to exit the PM,...

Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae) which are transmitted by phlebotomine sand flies (Diptera: Phlebotominae). For the dixenous life cycle, leishmania parasites are equipped with enzymes that facilitate survival in both insect vectors and mammalian hosts. Gene for the enzyme catalase which protects cells from reactive oxygen species by the elimination of H2O2 and is present in related monoxenous trypanosomatids is, however, missing in Leishmania genome. Chitinase can be involved in the interaction of leishmania parasites with chitin-containing structures in sand flies (peritrophic matrix, stomodeal valve). The expression of the enzyme in amastigotes suggests its significant function also in the mammalian host. I tested the role of these enzymes in the life cycle of leishmania by direct comparison of L. mexicana mutants (i) with inserted catalase gene and (ii) with deleted chitinase gene with control groups. I conducted experimental infections of Lu. longipalpis including transmission of leishmania to the hosts by bite, tested the survival of leishmania in macrophages and performed experimental infections of BALB/c mice followed by xenodiagnoses. The experiments confirmed that the presence of catalase in leishmania does...

Digestion of proteins in the midgut of lepidopteran larvae relies on different types of peptidases, among the trypsins and chymotrypsins. In this work four chymotrypsinlike peptidases (MsCTLP1–4) were identified from the larval midgut of M. sexta, which are distantly related to another chymotrypsin (MsCT), a previously described peptidase present in the larval midgut of M. sexta. MsCTLP1–4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. Examination of MsCTLP expression in different tissues showed that most of the peptidases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. Expression analysis of MsCTLPs at different physiological states revealed that the mRNA amounts did not differ considerably in feeding and starving larvae except for MsCTLP2, whose mRNA dropped significantly upon starvation. During molting, however, the mRNA amounts of all MsCTLPs dropped significantly. Immunological determination of MsCTLP1 amounts showed that the mature peptidase was only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or molting larvae, suggesting that MsCTLP1 secretion is suspended during starvation or molt. Differential regulation of transcript levels as well as their partial expression in Malpighian tubules might point to a role, which is distinct from digestion for at least some MsCTLPs. In line with this assumption, MsCTLP1 was shown to interact with the chitin synthase 2 (MsCHS2), necessary for chitin synthesis in the course of peritrophic matrix formation in the midgut of M. sexta. The occurrence of this interaction in vivo is supported by colocalization and co-immunoprecipitation. The data suggest that chitin synthesis is controlled by an intestinal proteolytic signaling cascade linking chitin synthase activity to the nutritional state of the larvae. As MsCTLP1 appears to be involved in such signaling cascades, other midgut peptidases could have other targets and may therefore regulate different activities. To gain more insight into the functions of CTLPs, the gene family encoding these peptidases in the genome of the red flour beetle, T. castaneum, was analyzed. Using an extended search pattern, 14 TcCTLP genes were identified that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. Analysis of the expression patterns of seven TcCTLP genes at various developmental stages revealed that some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A). Others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP- 6C/D/E). TcCTLP genes were expressed predominantly in the midgut where they presumably function in digestion. However, TcCTLP-5C and TcCTLP-6C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, western blots were performed using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6C suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, systemic RNAi experiments were performed. While injections of dsRNAs to TcCTLP-5A/B and TcCTLP-6A/D/E into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects. Recombinant expressed TcCTLP-5C2 was moreover activated by trypsin and was able to hydrolyze AAPF, hence making TcCTLP-5C the first described chymotrypsin-like peptidase ever to be involved in molting.