Search

12 pages, 7 figures.-- Availability of data and materials: Data are available from the corresponding author upon reasonable request., The midgut tract of decapods is a digestive organ involved in the synthesis of peritrophic membrane, food transport, absorption of nutrients, and osmoregulation. The midgut tract has been described in detail in adult decapods, but little information is available regarding the morphology and ultrastructure of the midgut tract in larval stages. The present study describes the midgut tract and the midgut–hindgut junction of the larvae of the common spider crab Maja brachydactyla Balss, 1922 using techniques that included dissection, light microscopy, and electron microscopy. The study is mainly focused on the stages of zoea I and megalopa. The results obtained in this study show that the larval midgut tract is a short and simple tube positioned anteriorly, between the stomach and the hindgut tract. During larval development, the maximum length of the midgut tract increases significantly, but no differences were found on either the maximum diameter or the morphological traits of the organ. The midgut tract is active at least ca. 12 h after hatching, as suggested by the presence of the peritrophic membrane in the lumen, the presence of abundant electro-dense vesicles in the cell apex, and the release of the vesicle content on the organ lumen. The midgut–hindgut junction forms an abrupt transition between the midgut tract and the hindgut tract in which epithelial cells with mixed features of midgut and hindgut do not occur, G.G.: INIA Project (grant number RTA2011-00004-00-00) funded by Ministerio de Economía y Competitividad (Spanish Ministry of Economy and Competitiveness). D.C.: FPI-INIA fellowship (INIA Project RTA2011-00004-00-00) funded by Ministerio de Economía y Competitividad (Spanish Ministry of Economy and Competitiveness)., With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Chitin deacetylase (CDA) can accelerate the conversion of chitin to chitosan, influencing the mechanical properties and permeability of the cuticle structures and the peritrophic membrane (PM) in insects. Putative Group V CDAs SeCDA6/7/8/9 (SeCDAs) were identified and characterized from beet armyworm Spodoptera exigua larvae. The cDNAs of SeCDAs contained open reading frames of 1164 bp, 1137 bp, 1158 bp and 1152 bp, respectively. The deduced protein sequences showed that SeCDAs are synthesized as preproteins of 387, 378, 385 and 383 amino acid residues, respectively. It was revealed via spatiotemporal expression analysis that SeCDAs were more abundant in the anterior region of the midgut. The SeCDAs were down-regulated after treatment with 20-hydroxyecdysone (20E). After treatment with a juvenile hormone analog (JHA), the expression of SeCDA6 and SeCDA8 was down-regulated; in contrast, the expression of SeCDA7 and SeCDA9 was up-regulated. After silencing SeCDAV (the conserved sequences of Group V CDAs) via RNA interference (RNAi), the layer of intestinal wall cells in the midgut became more compact and more evenly distributed. The vesicles in the midgut were small and more fragmented or disappeared after SeCDAs were silenced. Additionally, the PM structure was scarce, and the chitin microfilament structure was loose and chaotic. It was indicated in all of the above results that Group V CDAs are essential for the growth and structuring of the intestinal wall cell layer in the midgut of S. exigua. Additionally, the midgut tissue and the PM structure and composition were affected by Group V CDAs.

Spodoptera frugiperda is one of the world’s major agricultural pests and it occurs in many countries around the world. In Lepidoptera, the peritrophic membrane in the midgut protects the midgut epithelial cells and facilitates the digestion and absorption of food. Its main components are chitin and protein. Chitin synthase (CHS), as the last enzyme in the chitin biosynthesis pathway, is very important. Here, the expression of chitin synthase B (CHSB) gene identified from midgut was inhibited by RNA interference to explore its function in the metabolism of trehalose and chitin of S. frugiperda larvae. The results were as follows: SfCHSB was highly expressed in actively feeding larvae. Second, knockdown of SfCHSB resulted in decreased expression of most genes involved in chitin metabolism, including chitinase, and abnormal phenotypes of S. frugiperda were observed. In addition, membrane-bound trehalase activity and glucose content increased, and trehalose content decreased at 24 h after dsSfCHSB injection. Trehalase activities increased significantly and the glycogen content decreased at 48 h after dsSfCHSB injection. Finally, the activity of chitinase decreased and the content of chitin increased significantly. Our results indicate that SfCHSB expression is specific, SfCHSB has a large role in regulating chitin metabolism, and it has broad application prospects in the biocontrol of S. frugiperda.

Onchocerciasis is a neglected tropical disease (NTD) caused by Onchocerca Diesing 1841 (Spirurida: Onchocercidae) nematodes transmitted by blackflies. It is associated with poverty and imposes a significant health, welfare and economic burden on many tropical countries. Current methods to visualize infections within the vectors rely on invasive methods. However, using micro‐computed tomography techniques, without interference from physical tissue manipulation, we visualized in three dimensions for the first time an L1 larva of an Onchocerca species within the thoracic musculature of a blackfly, Simulium damnosum s.l. Theobald 1903 (Diptera: Simuliidae), naturally infected in Ghana. The possibility that thicker peritrophic membranes in savannah flies could account for their lower parasite loads was not supported, but there were limits to our analysis. While there were no statistically significant differences between the mean thicknesses of the peritrophic membranes, in the anterior, dorsal and ventral regions, of forest and savannah blackflies killed 34–48 min after a blood‐meal, the thickness of the peritrophic membrane in the posterior region could not be measured. Micro‐computed tomography has the potential to provide novel information on many other parasite/vector systems and impactful images for public engagement in health education., Micro‐computed tomography was used to visualize in 3D an L1 larva of an Onchocerca species within the thoracic musculature of a blackfly, Simulium damnosum s.l., naturally infected in Ghana.There were no statistically significant differences between the mean thicknesses of the peritrophic membranes of forest and savannah blackflies killed 34–48 min after a blood‐meal.Micro‐computed tomography has the potential to provide novel information on many other parasite/vector systems and impactful images for public engagement in health education.

The insect midgut secretes a semi-permeable, acellular peritrophic membrane (PM) that maintains intestinal structure, promotes digestion, and protects the midgut from food particles and pathogenic microorganisms. Peritrophin is an important PM protein (PMP) in the PM. Here, we identified 11 peritrophins with 1&ndash, 16 chitin binding domains (CBDs) comprising 50&ndash, 56 amino acid residues. Multiple CBDs in the same peritrophin clustered together, rather than by species. The CBD contained six highly conserved cysteine residues, with the key feature of amino acids between them being CX11-15CX5CX9-14CX11-12CX6-7C. Peritrophins with 2 and 4 CBDs (Bm09641 and Bm01504, respectively), and with 1, 8, and 16 CBDs (Bm11851, Bm00185, and Bm01491, respectively) were mainly expressed in the anterior midgut, and throughout the midgut, respectively. Survival rates of transgenic silkworms with Bm01504 overexpression (Bm01504-OE) and knockout (Bm01504-KO) infected with B. morinucleopolyhedrovirus (BmNPV) were significantly higher and lower, whereas expression of the key viral gene, p10, were lower and higher, respectively, compared with wild type (WT). Therefore, Bm01504-OE and Bm01504-KO transgenic silkworms were more and less resistant, respectively, to BmNPV. Bm01504 plays important roles in resisting BmNPV invasion. We provide a new perspective for studying PM function, and reveal how the silkworm midgut resists invasive exogenous pathogenic microorganisms.

Serine peptidases (SPs) are responsible for most primary protein digestion in Lepidoptera species. An expansion of the number of genes encoding trypsin and chymotrypsin enzymes and the ability to upregulate the expression of some of these genes in response to peptidase inhibitor (PI) ingestion have been associated with the adaptation of Noctuidae moths to herbivory. To investigate whether these gene family expansion events are common to other Lepidoptera groups, we searched for all genes encoding putative trypsin and chymotrypsin enzymes in 23 publicly available genomes from this taxon. Phylogenetic analysis showed that several gene family expansion events may have occurred in the taxon&rsquo, s evolutionary history and that these events gave rise to a very diverse group of enzymes, including proteins lacking the canonical SP catalytic triad. The expression profile of these enzymes along the midgut and the secretion mechanisms by which these enzymes enter the luminal content were also analyzed in Spodoptera frugiperda larvae using RNA-seq and proteomics. These results support the proposal of a midgut countercurrent flux responsible for the direction of these proteins to the anterior portion of the midgut and show that these enzymes reach the midgut lumen via both exocytosis and microapocrine secretion mechanisms.

Introduction and purpose: Leishmaniasis are neglected diseases that are endemic in more than half of the provinces of Iran. The causative agent of this disease is Leishmania protozoa and its transmission to human is through the bites of phlebotomine sand flies. This study reviews the growth and development of Leishmania in the body of sand flies at the molecular level. Methods: In this study a narrative review was carried out. The corpus was collected by searching the keywords of Leishmania, sand fly, phlebotomus, lutzomyia in the databases of PubMed, Scopus, SID, Web of Science, Ovid Medline, and Google Scholar search engine. The studied articles were those which has been published on the interaction between the sand fly vectors and Leishmania parasite until 2018. Results: The parasites grow when they encounter natural barriers in the body of the sand flies, including proteolytic enzymes, peritrophic membrane, excretion of the digestive system, and immune system in sand fly vectors. Attachment of Leishmania to sand fly digestive tract determines the ability of sand fly to support the development of parasite, which leads to the transmission of leishmaniasis.Conclusion: Studies on the development and life cycle of Leishmania within the body of sand flies are of utmost importance due to the spread of leishmaniasis in Iran and the world, difficulty in their control, and lack of effective vaccine products against leishmaniasis. The findings of the current study aimed at identifying and enhancing the natural barriers to the development of Leishmania in sand fly vectors and introducing new strategies for the control of leishmaniasis.

The Indianmeal moth, Plodia interpunctella, is one of the most damaging pests of stored products. We investigated the insecticidal properties of ApKTI, a Kunitz trypsin inhibitor from Adenanthera pavonina seeds, against P. interpunctella larvae through bioassays with artificial diet. ApKTI-fed larvae showed reduction of up to 88% on larval weight and 75% in survival. Trypsin enzymes extracted from P. interpunctella larvae were inhibited by ApKTI, which also demonstrated capacity to bind to chitin. Kinetic studies revealed a non-competitive inhibition mechanism of ApKTI for trypsin, which were further corroborated by molecular docking studies. Furthermore, we have demonstrated that ApKTI exhibits a hydrophobic pocket near the reactive site loop probably involved in chitin interactions. Taken together, these data suggested that the insecticidal activity of ApKTI for P. interpunctella larvae involves a dual and promiscuous mechanisms biding to two completely different targets. Both processes might impair the P. interpunctella larval digestive process, leading to larvae death before reaching the pupal stage. Further studies are encouraged using ApKTI as a biotechnological tool to control insect pests in field conditions.

The Cydia pomonella granulovirus (CpGV) GP37 has synergistic effects on the infectivity of nucleopolyhedroviruses (NPVs), however, the mechanism employed is unclear. In this study, in vitro and in vivo binding assays indicated that GP37 efficiently bound to the midgut peritrophic membrane (PM) of Spodoptera exigua larvae. Treatment with GP37 led to the damage of the PM&rsquo, s compacted structure and the generation of the PM perforations, and the enhancement of the PM&rsquo, s permeability. qPCR results further demonstrated that GP37 increased the ability of occlusion-derived virions (ODV) to cross the PM. R18-labeling experiments exhibited that GP37 also promoted the fusion of ODVs and insect midgut epithelia. Altogether, our present results revealed that the synergistic mechanism of GP37 to the infectivity of NPV might involve two parts. GP37 damaged the integrity of the PM after binding, which enhanced the PM&rsquo, s permeability and increased the ability of ODVs to cross the PM, finally facilitating the ODVs reaching the midgut. In addition, GP37 promoted the fusion of ODVs and insect midgut epithelia. Our data expand the understanding of the mechanism used by baculovirus synergistic factors and provide a foundation for the development of high-efficiency baculoviral insecticides.

Resumen La morfología, citología y algunos aspectos fisiológicos del tracto digestivo de larvas de cuarto estadio del siárido Bradysia impatiens se estudiaron utilizando microscopía de campo brillante. Se observó la división del tracto digestivo (estomodeo, mesenterón y proctodeo); las células que lo componen son columnares, regenerativas y endocrinas. Las larvas no presentan buche y la digestión del bolo alimenticio se realiza en el intestino medio; tienen dos ciegos gástricos bien desarrollados, que permiten suponer que existe una gran absorción de nutrientes. La membrana peritrófica es del tipo II, con un tamaño de poro que permite el paso de moléculas de hasta 150 kDa y divide el intestino medio en dos regiones, los espacios endo y ecto peritrófico. Tiene cuatro tubos de Malpighi. El intestino medio posee un gradiente de pH que va de alcalino en el intestino anterior a neutro en los ciegos gástricos y el intestino posterior. Tiene un mecanismo de recirculación que mejora la digestión y reutilización de enzimas digestivas, que va del espacio endoperitrófico al ectoperitrófico y viceversa. Las características morfofisiológicas mostradas en las larvas de B. impatiens permiten confirmar que son organismos bien adaptados como detritívoros. Además, la membrana peritrófica protege al intestino medio de agentes patógenos. Abstract The morphological, cytological and physiological aspects of the digestive tract of the siarid Bradysia impatiens fourth-instar larvae were studied using bright-field microscopy. The division of the digestive tract into the stomodeum (foregut), the mesenteron (midgut) and the proctodeum (hindgut) was observed; the second of which being composed of endocrine, regenerative, column-shaped cells. The larvae did not present a crop; hence digestion of the bolus occurs in the midgut. They have two well-developed gastric caeca, where it can be assumed that great nutrient absorption takes place. The peritrophic membrane is type II, with a pore size that allows the passage of molecules up to 150 kDa, and which divides the midgut into two regions: the endoperitrophic and ectoperitrophic spaces. There are four Malpighian tubules present. The midgut has a pH gradient ranging from mildly alkaline in the foregut to neutral in the caeca and hindgut. It has a recirculation mechanism stretching from the endoperitrophic to the ectoperitrophic space and vice versa, which improves digestion and optimizes enzyme production. The morphophysiological features observed in B. impatiens larvae confirm that these organisms are well-adapted detritivores. Additionally, the peritrophic membrane protects the midgut from pathogenic agents.

Background Mosquitoes transmit many vector-borne infectious diseases including malaria, dengue, chikungunya, yellow fever, filariasis, and Japanese encephalitis. The insecticidal δ-endotoxins Cry4, Cry11, and Cyt produced from Bacillus thuringiensis have been used for bio-control of mosquito larvae. Cry δ-endotoxins are synthesised as inactive protoxins in the form of crystalline inclusions in which they are processed to active toxins in larval midgut lumen. Previously, we demonstrated that the activated Cry4Ba toxin has to alter the permeability of the peritrophic membrane (PM), allowing toxin passage across PM to reach specific receptors on microvilli of larval midgut epithelial cells, where the toxin undergoes conformational changes, followed by membrane insertion and pore formation, resulting in larval death. A peritrophic membrane (PM)-binding calcofluor has been proposed to inhibit chitin formation and enhance baculovirus infection of lepidopteran Trichoplusia ni. Methods In this study, Aedes aegypti larvae were fed with the calcofluor and Cry4Ba toxin to investigate the effect of this agent on the toxicity of the Cry4Ba toxin. Results Calcofluor displayed an enhancing effect when co-fed with the Cry4Ba wild-type toxin. The agent could restore the killing activity of the partially active Cry4Ba mutant E417A/Y455A toward Ae. aegypti larvae. PM destruction was observed after larval challenge with calcofluor together with the toxin. Interestingly, calcofluor increased Cry4Ba toxin susceptibility toward semi-susceptible Culex quinquefasciatus larvae. However, calcofluor alone or in combination with the toxin showed no mortality effect on non-susceptible fresh-water fleas, Moina macrocopa. Conclusions Our results suggest that PM may contribute to the resistance of the mosquito larvae to Cry4Ba toxin. The PM-permeability alternating calcofluor might be a promising candidate for enhancing insect susceptibility, which will consequently improve Cry4Ba efficacy in field settings in the future.

Midgut cell and peritrophic membranes of grain pest Agrotis segetum larvae were examined by light microscope. Six biotinylated lectins were used in the assays by applying histochemical methods (Avidin-Biotin-Peroxidase). The aim of this study was to find lectins binding on peritrophic membranes if they were available as insecticidal agents. Results of the study indicated that lectins BPA (Bauhinia purpurea) and GS-I (Griffonia simplicifolia) strongly stained whereas, PNA (Pea Nut Agglutinin) and UEA-1 (Ulex europaeus) moderately stained the tissues. However, WGA (Wheat Germ Agglutinin) and Con-A (Canavalia ensiformis) stained the tissues weakly. The common feature of two binding proteins, BPA and GS-I lectin was binding to D-galactose. Our examinations revealed that D-galactose mostly exists in A. segetum larvae midgut cell membranes and peritrophic membrane and this means BPA and GS-I lectins can be used as insecticidal agents against A. segetum. © 2018, Gazi University Eti Mahallesi. All rights reserved.

The mid gut in insects is usually lined with a peritrophic membrane (PM), which protects it from microbial infections and physical contact with ingested food. Calcofluor is an optical brightener that has strong chitin-binding properties, which disrupt the chitin-protein structure of PM. In this study, we investigated the effects of calcofluor on the structure of the PM, activity of some common digestive enzymes and biological characteristics of the Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae). Twenty hours after feeding on a calcofluor-containing diet, the PM was completely eliminated. We recorded significant effects of calcofluor on the activity of digestive enzymes in the mid and hindguts of larvae. In both these regions of the gut, in particular, there were significant increases in the activity of α-amylase, α-galactosidase and trypsin. The activities of α-glucosidase and β-glucosidase in the midgut of treated larvae were lower than in the control, but were significantly higher in the hindgut of treated larvae. The larvae that fed on on calcofluor for 20 h, took longer to complete their larval and pupal stages, were lighter in weight and fewer reached maturity and laid fewer eggs. Results of this and previous studies highlight the crucial role of PM in enzyme recycling and digestion. PM disruption may negatively affect insect development on the one hand, by decreasing the efficiency of food digestion and, on the other, by increasing the costs associated with an increased enzyme demand and disrupted enzyme recycling mechanism.

The cabbage looper, Trichoplusia ni, a global generalist lepidopteran pest, has developed resistance to many synthetic and biological insecticides, requiring effective and environmentally acceptable alternatives. One possibility is the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). This baculovirus is highly infectious for T. ni, with potential as a biocontrol agent, however, its effectiveness is strongly influenced by dietary context. In this study, microscopy and transcriptomics were used to examine how the efficacy of this virus was affected when T. ni larvae were raised on different diets. Larvae raised on potato host plants had lower chitinase and chitin deacetylase transcript levels and thickened, multilayered peritrophic membranes than those reared on either cabbage or artificial diet. These changes help explain the significantly lower susceptibility of potato reared individuals to baculovirus, underlining the importance of considering the dietary influences on insect susceptibility to pathogens when applying biological control agents in integrated pest management strategies.

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae. [BMB Reports 2014; 47(10): 546-551]

Peritrophic membranes (PMs) are composed of chitin and protein. Chitin and protein play important roles in the structural formation and function of the PM. A new type I PM protein, HoCBP76, was identified from the Holotrichia oblita. HoCBP76 was shown as a 62.3 kDa protein by SDS-PAGE analysis and appeard to be associated with the PM throughout its entire length. In H. oblita larvae, the midgut is the only tissue where HoCBP76 could be detected during the feeding period of the larvae. The predicted amino acid sequence indicates that it contains seven tandem chitin binding domains belonging to the peritrophin-A family. HoCBP76 has chitin binding activity and is strongly associated with the PM. The HoCBP76 was not a mucin-like glycoprotein, and the consensus of conserved cysteines appeared to be CX13–17CX5CX9CX12CX7C. Western blot analysis showed that the abundance of HoCBP76 in the anterior, middle and posterior regions of the midgut was similar, indicating that HoCBP76 was secreted by the whole midgut epithelium, and confirmed the H. oblita PM belonged to the Type I PM. Immunolocalization analysis showed that HoCBP76 was mainly localized in the PM. The HoCBP76 is the first PM protein found in the H. oblita; however, its biochemical and physiological functions require further investigation.

The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. PM proteins are important determinants for PM structure and formation. In this study, the silkworm Bombyx mori midgut PM protein BmCDA7 was identified by proteomic tools. The full-length BmCDA7 cDNA is 1357 bp; the deduced protein is composed of 379 amino acid residues and includes a 16 amino acid residue signal peptide, a putative polysaccharide deacetylase-like domain and 15 cysteine residues present in three clusters. The heterologously expressed proteins of the BmCDA7 gene in yeast displayed chitin deacetylase activity. Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels. The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars. The functions and regulatory mechanism of BmCDA7, however, need further investigation.

The Indianmeal moth, Plodia interpunctella, is one of the most damaging pests of stored products. We investigated the insecticidal properties of ApKTI, a Kunitz trypsin inhibitor from Adenanthera pavonina seeds, against P. interpunctella larvae through bioassays with artificial diet. ApKTI-fed larvae showed reduction of up to 88% on larval weight and 75% in survival. Trypsin enzymes extracted from P. interpunctella larvae were inhibited by ApKTI, which also demonstrated capacity to bind to chitin. Kinetic studies revealed a non-competitive inhibition mechanism of ApKTI for trypsin, which were further corroborated by molecular docking studies. Furthermore, we have demonstrated that ApKTI exhibits a hydrophobic pocket near the reactive site loop probably involved in chitin interactions. Taken together, these data suggested that the insecticidal activity of ApKTI for P. interpunctella larvae involves a dual and promiscuous mechanisms biding to two completely different targets. Both processes might impair the P. interpunctella larval digestive process, leading to larvae death before reaching the pupal stage. Further studies are encouraged using ApKTI as a biotechnological tool to control insect pests in field conditions.

The bean shoot borer Epinotia aporema Wals. (Lepidoptera: Tortricidae) and the velvet bean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) are key pests of soybean and other legume crops in South America. They are often found simultaneously in certain regions. A. gemmatalis nucleopolyhedrovirus (AgMNPV) is widely used to control A. gemmatalis. More recently, E. aporema granulovirus (EpapGV) has been characterized and evaluated as a bioinsecticide for E. aporema. In order to increase its potential use and to design optimized strategies for the management of lepidopteran pests, we evaluated the interaction between EpapGV and AgMNPV on third instar A. gemmatalis larvae. Larvae fed with 50 AgMNPV OBs/larva showed an increase in the mortality rates (from 42% to 81%) and a decrease in the median survival time (from 7.7 days to 5.7 days) when these OBs were mixed with 6000 EpapGV OBs/larva. When 300 AgMNPV OBs/larva were used alone or in combination with EpapGV OBs no changes in biological parameters were observed. No mortality was detected in A. gemmatalis larvae treated with EpapGV alone. In larvae fed with the viral mixtures, only AgMNPV DNA was detected by PCR. A. gemmatalis peritrophic membranes (PMs) examined by SDS–PAGE and scanning electron microscopy showed signs of damage. Notably, we found the presence of spheroidal bodies associated with damaged areas in the PMs of larvae fed with EpapGV but not in those that were given AgMNPV alone. These results show that EpapGV increases the viral potency of AgMNPV, and thus the insecticidal efficiency, suggesting that the use of formulations including both viruses might be a valuable tool for pest management. Fil: Biedma, Marina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Salvador, Ricardo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Ferrelli, Maria Leticia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Sciocco Cap, Alicia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Romanowski, Victor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina

Sanjaya, Machmudin D, Kurniawati ND. 2010. Kajian histologis infeksi SlNPV terhadap berat badan dan kerusakan membran peritrofik larva Spodoptera litura. Nusantara Bioscience 2: 135-140. Pengaruh infeksi SlNPV pada berat badan dan kerusakan membran peritrofik larva Spodoptera litura Fab. telah dilakukan. Metode yang digunakan adalah analisis probit, dan berdasarkan LD 50 virus yang terinfeksi untuk mengetahui berat badan dan kerusakan pasca infeksi. Kerusakan struktur histologi yang disebabkan oleh infeksi SlNPV (0, 315, 390, 465, 540 dan 615 PIB/mL) diamati setelah 0, 12, 24, 72 dan 96 jam pasca infeksi. Preparasi histologis dibuat dengan metode parafin setelah fiksasi dengan larutan Bouin, kemudian diiris setebal 7 um dan diwarnai dengan Hematoxilin- Eosin. Hasil penelitian menunjukkan bahwa paparan SlNPV menyebabkan penurunan konsumsi pangan terutama pada 540 PIB/mL dengan rata-rata 0.1675 mg. Pengamatan deskriptif pada struktur histologi membran peritrophic yang terkena infeksi SlNPV menunjukkan kecenderungan kerusakan, sementara pada kontrol tidak ada kerusakan sama sekali. Semakin lama paparan virion di dalam lumen midgut maka semakin tinggi terjadinya kerusakan membran peritrofik. Kerusakan paling parah terjadi 96 jam setelah infeksi. Hasilnya membuktikan bahwa virion haNPV dapat menghancurkan struktur histologi midgut. Kata kunci: SlNPV, Spodoptera litura, LD50, tingkat konsumsi, membran peritrophic.

A bioinformatics-based investigation of three insect species with completed genome sequences has revealed that insect chitinase-like proteins (glycosylhydrolase family 18) are encoded by a rather large and diverse group of genes. We identified 16, 16 and 13 putative chitinase-like genes in the genomic databases of the red flour beetle, Tribolium castaneum, the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, respectively. Chitinase-like proteins encoded by this gene family were classified into five groups based on phylogenetic analyses. Group I chitinases are secreted proteins that are the most abundant such enzymes in molting fluid and/or integument, and represent the prototype enzyme of the family, with a single copy each of the catalytic domain and chitin-binding domain (ChBD) connected by an S/T-rich linker polypeptide. Group II chitinases are unusually larger-sized secreted proteins that contain multiple catalytic domains and ChBDs. Group III chitinases contain two catalytic domains and are predicted to be membrane-anchored proteins. Group IV chitinases are the most divergent. They usually lack a ChBD and/or an S/T-rich linker domain, and are known or predicted to be secreted proteins found in gut or fat body. Group V proteins include the putative chitinase-like imaginal disc growth factors (IDGFs). In each of the three insect genomes, multiple genes encode group IV and group V chitinase-like proteins. In contrast, groups I–III are each represented by only a singe gene in each species.

Background Ecological studies routinely show genotype-genotype interactions between insects and their parasites. The mechanisms behind these interactions are not clearly understood. Using the bumblebee Bombus terrestris/trypanosome Crithidia bombi model system (two bumblebee colonies by two Crithidia strains), we have carried out a transcriptome-wide analysis of gene expression and alternative splicing in bees during C. bombi infection. We have performed four analyses, 1) comparing gene expression in infected and non-infected bees 24 hours after infection by Crithidia bombi, 2) comparing expression at 24 and 48 hours after C. bombi infection, 3) determining the differential gene expression associated with the bumblebee-Crithidia genotype-genotype interaction at 24 hours after infection and 4) determining the alternative splicing associated with the bumblebee-Crithidia genotype-genotype interaction at 24 hours post infection. Results We found a large number of genes differentially regulated related to numerous canonical immune pathways. These genes include receptors, signaling pathways and effectors. We discovered a possible interaction between the peritrophic membrane and the insect immune system in defense against Crithidia. Most interestingly, we found differential expression and alternative splicing of immunoglobulin related genes (Dscam and Twitchin) are associated with the genotype-genotype interactions of the given bumblebee colony and Crithidia strain. Conclusions In this paper we have shown that the expression and alternative splicing of immune genes is associated with specific interactions between different host and parasite genotypes in this bumblebee/trypanosome model., BMC Genomics, 15, ISSN:1471-2164

Peritrophic membranes (PMs) are composed of proteins, proteoglycans and chitin that play important roles in the structural formation and function of the PM. This study identified and characterized a new chitin binding protein named HpCBP45 by immunoscreening of the Holotrichia parallela larvae midgut expression library. The predicted amino acid sequence indicates that it contains eight tandem chitin binding domains belonging to the peritrophin-A family. The HpCBP45 protein was expressed as a recombinant protein in the yeast Pichia pastoris and chitin binding assay demonstrated that recombinant HpCBP45 protein could strongly bind to chitin. qRT-PCR analysis showed that HpCBP45 was mainly localized in the midgut, further confirming the H. parallela PM belongs to Type I PM. The discovery and characterization of the peritrophic membrane protein HpCBP45 provides a basis for the further investigation of its biochemical and physiological functions in H. parallela.

The main function of baculoviral chitinase protein (V-CHIA) is to promote the final liquefaction of infected host larvae, facilitating the dispersion of occlusion bodies (OBs) in the environment. In this study, a v-chiA from Epinotia aporema Granulovirus (EpapGV) was identified and characterized. The 1,713 base pairs long open reading frame encodes a protein of 570 amino acids with a predicted molecular weight of 63 kDa. EpapGV V-CHIA sequence alignment resulted 62 % identical to Pieris rapae GV and Blastp search revealed a high conservation among all baculovirus chitinases. Amino acid sequence analysis indicated that the C-terminal KDEL present in most alphabaculovirus chitinases is absent in EpapGV V-CHIA, as well as in the rest of the betabaculoviruses. Phylogenetic analysis was performed with bacterial, lepidopteran, and baculoviral chitinase sequences available in databases. Using an AcMNPV bacmid (bApGOZA) a recombinant Ac-chiAEpapGV was obtained in order to overexpress EpapGV V-CHIA in cell culture. The presence of chitinase was detected in purified AcMNPV-chiAEpapGV OBs. Peritrophic membranes of Anticarsia gemmatalis larvae fed with recombinant OBs showed an altered structure. The results presented in this study show that EpapGV chitinase overexpression in recombinant baculovirus can cause association of this protein with OBs, and suggest that this could be used to evaluate the protein role in early stages of baculoviral infections., Instituto de Biotecnología y Biología Molecular

The glycoprotein Cb-peritrophin-42 was localized in third instar larvae of Chrysomya bezziana using primary antibodies raised against a non-glycosylated bacterial recombinant form of this protein. Both immunofluorescent antibody techniques on unfixed whole mounts of gut tissues and immunogold electron microscopy techniques on ultra-thin sections of fixed and embedded tissues were employed. The protein was shown to be exposed over the whole of both surfaces of the peritrophic membrane and to occur throughout its thickness. Immunogold labelling indicated that Cb-peritrophin-42 was expressed in the peritrophic membrane-secreting cells of the cardia, a specialized peritrophic membrane-forming organ situated at the junction of the foregut and midgut. The accessibility of Cb-peritrophin-42 present in intact peritrophic membrane to the primary antibodies used in the immunofluorescent antibody localization indicates that this glycoprotein is a potential molecular target for vaccination of host animals against larvae of Chrysomya bezziana.

sources of potential protective antigens from the sheep blowfly Lucilia cuprina. Their importance in the screwworm fly Chrysomya bezziana has now been investigated. Purified serine proteases from Chrysomya bezziana were tested for their potential as vaccine antigens in sheep, efficacy being assessed by in vitro and in vivo assays with larval Chrysomya bezziana. No effect of vaccination was observed by the in vitro assay. However, in the in vivo challenge, larval weights were diminished in the vaccinated sheep, although larval recoveries increased marginally. Vaccination with Chrysomya bezziana peritrophic membrane does induce an effective immune response against the parasite resulting in a significant reduction in larval growth and considerable larval mortality in the in vitro assay. Sequential fractionation of the peritrophic membrane with various surfactants and chaotrophic agents of increasing solubilisation capacity resulted in the separation of discrete groups of proteins. The groups of fractionated proteins were tested in a vaccination trial in sheep with vaccine efficacy assessed by in vitro assays. The urea extract, guanidine-HCl extract and SDS soluble fraction each induced significant levels of protection against Chrysomya bezziana larvae but the effects were poorer than those obtained from vaccination with whole, native peritrophic membrane. Several major proteins selected from the three most protective fractions were purified by SDS polyacrylamide gel electrophoresis. Since insufficient quantities of these proteins were available for vaccination trials, they were either sequenced directly from the N-terminus or subjected to endoproteinase Lys-C digestion, followed by peptide purification and amino acid sequencing. This gave the information necessary for the expression of several of these roteins as recombinants in a form suitable for vaccination studies.

Three candidate antigens, Chrysomya bezziana peritrophin-48, Chrysomya bezziana peritrophin-15 and Chrysomya bezziana peritrophin-42, were prepared for recombinant protein production in Escherichia coli using a variety of expression vectors. Cb peritrophin-48 was expressed as a recombinant protein possessing a carboxy-terminal hexaHis tag. Cb peritrophin-15 was expressed as both a glutathione S-transferase fusion protein and as an amino-terminal hexaHis tagged protein. The glutathione Stransferase Cb peritrophin-15 construct produced a heterogeneous group of fusion proteins. Cb peritrophin-42 was also expressed as an amino-terminal hexaHis tagged protein. The two putative domains of Cb peritrophin-42 were also separately expressed, again with amino-terminal hexaHis tags. Cultures of the hexaHis constructs Cb peritrophin-48, -15 and –42 were demonstrated to be useful for the production and purification of these protein antigens and were scaled-up for vaccine trials and protein characterization studies.

The construction and use of cDNA libraries for the isolation of genes encoding candidate antigens for use in a recombinant vaccine against Chrysomya bezziana is described. RNA was isolated and mRNA purified from first and third instar larvae of Chrysomya bezziana and used in the synthesis of two cDNA libraries in the bacteriophage vector λ ZAP express®. These libraries were screened using Digoxigenin-labeled DNA probes obtained from two independent approaches. First, a homolog approach used probes designed from previously characterized peritrophic membrane genes identified from the related myiasis fly, Lucilia cuprina. Secondly, a de novo approach used amino-terminal and internal peptide sequence information derived from purified Chrysomya bezziana peritrophic membrane proteins to generate DNA probes. Three peritrophic membrane genes were identified and characterized. Chrysomya bezziana peritrophin-48 was identified using the homolog approach and, Chrysomya bezziana peritrophin-15 and Chrysomya bezziana peritrophin-42 were identified using the de novo approach. The identification of these genes as encoding candidate antigens against Chrysomya bezziana has allowed the production of recombinant proteins for use in vaccination trials

Densoviruses are parvoviruses that can be lethal for insects of different orders at larval stages. Although the horizontal transmission mechanisms are poorly known, densoviral pathogenesis usually starts with the ingestion of contaminated food by the host. Depending on the virus, this leads to replication restricted to the midgut or excluding it. In both cases the success of infection depends on the virus capacity to enter the intestinal epithelium. Using the Junonia coenia densovirus (JcDNV) as the prototype virus and the lepidopteran host Spodoptera frugiperda as an interaction model, we focused on the early mechanisms of infection during which JcDNV crosses the intestinal epithelium to reach and replicate in underlying target tissues. We studied the kinetics of interaction of JcDNV with the midgut epithelium and the transport mechanisms involved. Using several approaches, in vivo , ex vivo , and in vitro , at molecular and cellular levels, we show that JcDNV is specifically internalized by endocytosis in absorptive cells and then crosses the epithelium by transcytosis. As a consequence, viral entry disturbs the midgut function. Finally, we showed that four mutations on the capsid of JcDNV affect specific recognition by the epithelial cells but not their binding.

The Bacillus cereus sensu lato complex has recently been divided into several phylogenetic groups with clear differences in growth temperature range. However, only a few studies have investigated the actual pathogenic potential of the psychrotolerant strains of the B. cereus group at low temperature, and little information is available concerning gene expression at low temperature. We found that vegetative cells of the psychrotolerant B. weihenstephanensis strain KBAB4 were pathogenic against the model insect Galleria mellonella at 15°C but not at 30°C. A similar temperature-dependent difference also was observed for the supernatant, which was cytotoxic to Vero epithelial cell lines and to murine macrophage J774 cells at 15°C but not at 30°C. We therefore determined the effect of low temperature on the production of various proteins putatively involved in virulence using two-dimensional protein gel electrophoresis, and we showed that the production of the Hbl enterotoxin and of two proteases, NprB and NprP2, was greater at a growth temperature of 15°C than at 30°C. The quantification of the mRNA levels for these virulence genes by real-time quantitative PCR at both temperatures showed that there was also more mRNA present at 15°C than at 30°C. We also found that at 15°C, hbl mRNA levels were maximal in the mid- to late exponential growth phase. In conclusion, we found that the higher virulence of the B. cereus KBAB4 strain at low temperature was accompanied by higher levels of the production of various known PlcR-controlled virulence factors and by a higher transcriptional activity of the corresponding genes.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Plodia interpunctella (Indian meal moth) is a cosmopolitan pest that attacks not only a wide range of stored grain as well other food products. Due to its economic importance several researches have focused in a method with ability to control this pest with few or no damage to the environment. The study of digestive enzymes inhibitors, lectins and chitin-binding proteins, has often been proposed as an alternative to reduce insect damage. In this study we report the major classes of digestive enzymes during larval growth in P. Interpunctella, being those proteinases actives at pH 9.5 and optimum temperature of 50 oC to both larvae of the 3rd instar and pre-pupal stage of development. In vitro and zymogram assays presented the effects of several inhibitors, such as SBTI, TLCK and PMSF to intestinal homogenate of 3rd instar larvae of 62%, 92% and 87% of inhibition and In pre-pupal stage of 87%, 62 % and 55% of inhibition, respectively. Zymograms showed inhibition of two low molecular masses protein bands by TLCK and that in presence of SBTI were retarded. These results are indicative of predominance of digestive serine proteinases in gut homogenate from Plodia interpunctella larvae. This serine proteinase was then used as a target to evaluate the effect of SBTI on larvae in in vivo assay. Effect of SBTI on mortality and larval mass was not observed at until 4% of concentration (w/w) in diets. Chitin, another target to insecticidal proteins, was observed by chemical method. Moreover, optic microscopy confirmed the presence of a peritrophic membrane. Established this target, in vivo effect of EvV, a chitin binding vicilin, evaluated during the larval development of P. interpunctella and was obtained a LD50 of 0,23% and WD50 of 0,27% to this protein. Mechanism of action was proposed through of the in vivo digestibility of EvV methodology. During the passage through the larval digestive tract was observed that EvV was susceptible to digestive enzymes and a reactive fragment, visualized by Western blotting, produced by digestion was recovered after dissociation of the peritrophic membrane. The bound of EvV to peritrophic membrane was confirmed by immunohystochemical assays that showed strong immunofluorescent signal of EvV-FITC binding and peritrophic membrane. These results are a indicative that vicilins could be utilized as potential insecticide to Plodia interpunctella and a control methods using EvV as bioinsecticide should be studied to reduce lost caused by storage insect pests Plodia interpunctella (traça-indiana-da-farinha) é uma praga cosmopolita que ataca não somente uma ampla gama de produtos armazenados, mas também outros produtos alimentícios. Devido a sua importância econômica várias pesquisas têm sido realizadas com o intuito de identificar um método capaz de controlar esta praga sem danos ao ambiente. O estudo de inibidores de enzimas digestivas, lectinas e proteínas que se ligam à quitina tem sido proposto como uma alternativa para controlar o dano causado por estes insetos. Neste estudo alvos específicos para inibidores de enzimas e proteínas ligantes à quitina foram identificados nas larvas desta praga. Para isso, durante o desenvolvimento de larvas de P. interpunctella as classes de enzimas digestivas alvos foram identificadas por ensaios de atividade in vitro e SDS-PAGE, pH e temperatura ótimos avaliados para a indicação de possíveis proteínas inibidoras para a principal classe de proteinase detectadas no intestino das larvas. Outro alvo para proteínas deletérias foi indicado pela identificação da membrana peritrófica por ensaios químicos de detecção de quitina e por microscopia de luz. Durante o período de desenvolvimento as larvas de P. interpunctella, alimentadas com uma dieta baseada em bagaço de cana, passaram por 5 ínstares e pelo estágio pré-pupal. A maior atividade proteolítica (UA/intestino) foi detectada no estágio pré-pupal, enquanto que a maior atividade proteolítica específica (UA/mg proteína) foi observada no terceiro ínstar, utilizando azocaseína como substrato a pH 9,5 e a 50°C. A inibição das proteinases presentes no homogenato intestinal de larvas de terceiro ínstar foi mais evidente quando inibidores de proteinases serínicas (SBTI, TLCK e PMSF, com 96%, 89% e 20% de inibição, respectivamente) foram utilizados nos ensaios. No estágio pré-pupal, a maior inibição observada foi com SBTI (96%), TLCK (81 %) e TPCK (20%), indicando a predominância de atividade enzimática de proteinases serínicas a pH 9,5 no intestino de Plodia interpunctella. Por zimograma foi observada inibição de bandas de menor massa molecular por TLCK e um atraso na corrida eletroforética dessas bandas causado por SBTI. Quando avaliado o efeito in vivo de SBTI no desenvolvimento larval, não foi observada mortalidade e nem efeito na massa das larvas sobreviventes. Estabelecido o segundo alvo de atuação, baseado na ligação à quitina, bioensaios usando a vicilina EvV foram realizados, onde um LD50 de 0,23% e um WD50 de 0,27% foram estabelecidos para esta proteína deletéria. O mecanismo de ação foi verificado por ensaios de digestibilidade de EvV durante a passagem pelo trato intestinal larval, sendo observado o envolvimento de um fragmento reativo, observado por imunodetecção, no efeito deletério da vicilina. A ligação de EvV à membrana peritrófica foi comprovada através de ensaios de imunohistoquímica. Estes resultados apontam para uma vicilina ligante à quitina que pode vir a ser utilizada como bioinseticida para Plodia interpunctella

Bombyx mori, hybrid Shunrei x Shogetsu, is susceptible to Cry1Aa and insensitive to Cry1Ac. Toxicity of Cry toxin can be correlated with the presence of a specific receptor in midgut epithelial cell membrane in insect. In surface plasmon resonance (SPR) and ligand blot analysis, however, many kinds of brush border membrane vesicle (BBMV) proteins were shown to bind to Cry1Ac with almost equal intensity in both resistance and susceptible insects. This suggests that majority of the bindings between Cry1Ac and BBM proteins of B. mori may be pseudo-binding. Therefore, we have hypothesized that these pseudo-bindings to Cry1Ac should not lead to insect death. Cry1A must pass through the peritrophic membrane (PM) before getting to BBM, thus PM plays a vital role for the action of Cry1A toxin as well as BBM. To elucidate the Cry1Ac insusceptibility of B. mori, we searched Cry1Ac binding proteins which are involved in the pseudo-binding in PM and BBM of B. mori. Here, we present the evidences showing some PM and BBM proteins can act as pseudo-receptor for Cry1Ac in B. mori. First of all, we evaluated permeability of Cry1Aa or Cry1Ac through the PM. An apparatus for the estimation of passing through PM was constructed, then BSA (66kDa), Carbonic anhydrase (29 kDa) and Cry1Aa were passed through the PM at 0.37 μg/mm2PM/hr. These passages seemed to be a diffusion because the rate was almost same, even though molecular size was different from each other. In contrast, Cry1Ac didn't pass through the PM for the first 2 hrs. However, during 3rdhr, Cry1Ac passed through the PM at 0.34 μg/mm2PM/hr, which may be due to the destruction of the PM or the saturation of Cry1Ac binding site on the PM. The Cry1Ac trapping during the first 2 hrs can be closely correlated with the interaction of toxins to some PM proteins. We performed ligand blot analysis of Cry1A toxins with PM proteins. As expected, Cry1Ac binding proteins were detected in detergent soluble fractions. Particularly, 125 (P125), 100 (P100), 95 (P95) and 55 (P55) kDa proteins, from class 2 which were obtained with 1% Triton X-100 and 165 (PI65) kDa proteins of class 3 which was extracted with 2% SDS and 2% mercaptoethanol, were shown to bind to Cry1Ac only but not Cry1Aa. These results suggested the prevention of Cry1Ac from passing through the PM happens through the trapping of PM proteins. Cry1Ac recognizes N-acetylgalactosamine (GalNAc) occurring as sugar side chain of proteins localizing in BBM. Cry1Ac trapping by the whole class 2 and 3 PM proteins had 70% inhibition in the presence of GalNAc, but Cry1Aa trapping was not inhibited., In ligand blot, the bindings between Cry1Ac with P125, P95, P55 and P165 were almost completely inhibited by GalNAc. We estimated the GalNAc effect on the Cry1Ac passage through the PM and co-inoculation of Cry1Ac and GalNAc was shown to facilitate the Cry1Ac passage through the PM with the rate of 0.45 μg/mm2PM/Hr. GalNAc residue of PM protein seemed to be involved in Cry1Ac trapping. Secondly, we also detected many kinds of Cry1Ac binding proteins from B. mori whole BBM protein, Triton X-100-soluble and -insoluble BBMV proteins. Although Cry1Ac has no toxicity to B. mori, many kinds of Cry1Ac binding proteins, such as 252, 115, 105, 100, 96 and 75 kDa, were detected. DEAE chromatography of Triton X-100-soluble BBMV proteins from the midgut revealed 96-kDa aminopeptidase that bound to Cry1Ac. The enzyme was purified to homogeneity and estimated to be a 96.4 kDa molecule on a silver-stained SDS-PAGE gel. However, the native protein was eluted as a single peak corresponding to ～190 kDa on gel filtration and gave a single band on native PAGE. The enzyme was determined to be an aminopeptidase N (APN96) from its substrate specificity. Antiserum to class 3 B. mori APN (BmAPN3) recognized APN96, but peptide mass fingerprinting revealed that 54% of amino the acids of matched peptides were identical to those of BmAPN3, suggesting that APN96 was a novel isoform of the APN3 family. On ligand blots, APN96 bound to Cry1Ac but not Cry1Aa or Cry1Ab, and the interaction was inhibited by GalNAc. The KD value of the APN96-Cry1Ac interaction was determined to be 1.83± 0.95μM. The lectin binding assay suggested that APN96 had an N-linked oligosaccharide or an O-linked mucin type one. The role of APN96 and other Cry1Ac binding proteins were discussed in relation to the insensitivity of B. mori to Cry1Ac. We believe that majority of these Cry1Ac binding proteins from both PM and BBM may act as pseudo-receptors to quench the toxicity of Cry1Ac, and propose here the model of mechanism for detoxification of Cry1Ac in B. mori. Activated Cry1Ac is initially trapped by PM proteins. In case, some Cry1Ac molecules pass through the PM and bind to BBM proteins, such as APN96, P252, 100, 105 and 75 kDa proteins. The bindings with these proteins reduce further the toxicity of Cry1Ac. It was reported that both the cadherin like protein and APN were shown to be related with Cry1Ab toxicity in Manduca sexta. We guess, however, there is no such involvement of these proteins in CrylAc binding in B. mori. CrylAc must bind to PM and/or BBMV proteins and stay there for a while without modification, thus the Cry1Ac can not form pores on BBMV., 新大院博（学）甲第167号

Made available in DSpace on 2022-04-29T08:42:49Z (GMT). No. of bitstreams: 0 Previous issue date: 1996-12-01 Ultrastructure of the anterior end of the midgut of three Neotropical stingless bees, including an obligate necrophagous species Trigona hypogea, suggests that this midgut region has no role in nutrient absorption because its lumen is reduced by a thick cuticle that lines the outer epithelium of the cardiac valve. A possible involvement of this midgut region in the production of the peritrophic membrane compounds is discussed. Departamento de Biologia Institute de Biociências Campus de Rio Claro, Cx. Postal 199, CEP 13506-900, Rio Claro, SP

Midgut glycoproteins of the malaria vector Anopheles tessellatus were partially characterised by gel electrophoresis and lectin binding. Specific binding to wheat germ agglutinin (WGA) and Concanavalin A (Con A) indicated the presence of N-linked core oligosaccharides in many proteins. Rabbit antibodies were produced against wheat germ agglutinin binding proteins (WGABP). These antibodies also recognised distinct proteins in the peritrophic membrane which is secreted into the midgut to enclose a bloodmeal. Rabbit anti-WGABP antibodies ingested in a bloodmeal containing infective gametocytes of the human malaria parasites Plasmodium falciparum and P. vivax tended to reduce infectivity of the parasites to vector mosquitoes. Chitotriose added to a bloodmeal also inhibited parasite development in the mosquito. The results are consistent with a hypothesis that N-acetyl glucosamine residues in mosquito midgut glycoproteins and/or midgut chitin and proteoglycan function as recognition sites for malaria parasites.