Midgut Wall Research Articles

The anatomy and histology of the midgut and Malpighian tubules ofCopris felschei Reitter, 1892 (Coleoptera: Scarabaeidae).

Copris are part of the Scarabaeidae family of Coleoptera. Copris are dung beetles or coprophagous beetles. These insects are called tunnelers because they excavate channels in the substrate. They use dead organisms and non-living organic compounds as a nutrient source. By breaking down dead matter, they provide nutrients that are important to the environment and necessary for the survival of other organisms. No studies have yet examined the midgut structure and Malpighian tubules of Copris. Therefore, this study investigated the histo-anatomical structure of the midgut and Malpighian tubules of Copris felschei Reitter, 1892 (Coleoptera: Scarabaeidae) using light and scanning electron microscopy (SEM) in detail. The midgut of C. felschei represents the largest part of the alimentary canal. Muscle layers and a monolayer of cylindrical epithelium surround the midgut wall. A peritrophic membrane envelops food in the midgut lumen, and crystals were observed within the lumen. The surface of the midgut has regenerative crypts and tracheae. The Malpighian tubules are arranged in two pairs and connect proximally between the midgut and hindgut. The Malpighian tubules are composed of a single layer of cuboidal epithelium. Numerous balloon-like tracheae were observed interspersed between the midgut and Malpighian tubules. Light and SEM images of the tracheae reveal a spongy structure with hollow chambers. These findings are anticipated to advance future research and deepen our understanding of the alimentary canal in Coleoptera, particularly within the Scarabaeidae family.

MiR-PC-3p-241582_34 Contributes to the Infection of Athetis lepigone by Regulating the Expression of HSWP4 in Nosema bombycis.

Athetis lepigone is a recurring pest in the maize seedling stage under the wheat-maize no-tillage direct seeding system in China's summer maize region. Our previous research identified a highly pathogenic Nosema bombycis to A. lepigone, which spore wall protein plays an important role in the infection process. However, the regulatory mechanism of this spore wall protein is still unclear. In this study, we explored the regulatory mechanism of miRNAs on spore wall proteins. Transcriptome sequencing results showed that expression of the spore wall protein, HSWP4, significantly increased in the germination group compared to dormancy group. Silencing of HSWP4 reduced the number of microsporidian spores breaking through the midgut wall cells of A. lepigone. Association analysis of small RNA and mRNA revealed that the targeting site of miR-PC-3p-241582_34 on HSWP4 was located in the CDS region, and miR-PC-3p-241582_34 had a significant negative regulatory relationship with HSWP4. The dual luciferase reporter assay demonstrated that miR-PC-3p-241582_34 significantly affected the luciferase activity of the HSWP4-3'UTR expression vector (P < 0.05). Delivery of miRNA mimics decreased the expression of HSWP4 and inhibited the behavior of microsporidian spores breaking through the midgut wall of A. lepigone. On the other hand, delivery of inhibitors produced opposite results, indicating that the miR-HSWP4 pathway plays an important role in microsporidian infection of A. lepigone. This study provides a new theoretical basis for understanding the pathogenic mechanism and gene regulation of microsporidia, as well as for the green control of A. lepigone.

In Vivo and In Vitro Interactions between Exopolysaccharides from Bacillus thuringensis HD270 and Vip3Aa11 Protein.

Bacillus thuringiensis (Bt) secretes the nutritional insecticidal protein Vip3Aa11, which exhibits high toxicity against the fall armyworm (Spodoptera frugiperda). The Bt HD270 extracellular polysaccharide (EPS) enhances the toxicity of Vip3Aa11 protoxin against S. frugiperda by enhancing the attachment of brush border membrane vesicles (BBMVs). However, how EPS-HD270 interacts with Vip3Aa11 protoxin in vivo and the effect of EPS-HD270 on the toxicity of activated Vip3Aa11 toxin are not yet clear. Our results indicated that there is an interaction between mannose, a monosaccharide that composes EPS-HD270, and Vip3Aa11 protoxin, with a dissociation constant of Kd = 16.75 ± 0.95 mmol/L. When EPS-HD270 and Vip3Aa11 protoxin were simultaneously fed to third-instar larvae, laser confocal microscopy observations revealed the co-localization of the two compounds near the midgut wall, which aggravated the damage to BBMVs. EPS-HD270 did not have a synergistic insecticidal effect on the activated Vip3Aa11 protein against S. frugiperda. The activated Vip3Aa11 toxin demonstrated a significantly reduced binding capacity (548.73 ± 82.87 nmol/L) towards EPS-HD270 in comparison to the protoxin (34.96 ± 9.00 nmol/L). Furthermore, this activation diminished the affinity of EPS-HD270 for BBMVs. This study provides important evidence for further elucidating the synergistic insecticidal mechanism between extracellular polysaccharides and Vip3Aa11 protein both in vivo and in vitro.

Interaction of acetamiprid, Varroa destructor, and Nosema ceranae in honey bees

Health of honey bees is threatened by a variety of stressors, including pesticides and parasites. Here, we investigated effects of acetamiprid, Varroa destructor, and Nosema ceranae, which act either alone or in combination. Our results suggested that interaction between the three factors was additive, with survival risk increasing as the number of stressors increased. Although exposure to 150 μg/L acetamiprid alone did not negatively impact honey bee survival, it caused severe damage to midgut tissue. Among the three stressors, V. destructor posed the greatest threat to honey bee survival, and N. ceranae exacerbated intestinal damage and increased thickness of the midgut wall. Transcriptomic analysis indicated that different combinations of stressors elicited specific gene expression responses in honey bees, and genes involved in energy metabolism, immunity, and detoxification were altered in response to multiple stressor combinations. Additionally, genes associated with Toll and Imd signalling, tyrosine metabolism, and phototransduction pathway were significantly suppressed in response to different combinations of multiple stressors. This study enhances our understanding of the adaptation mechanisms to multiple stressors and aids in development of suitable protective measures for honey bees. Environmental ImplicationWe believe our study is environmentally relevant for the following reasons: This study investigates combined effects of pesticide, Varroa destructor, and Nosema ceranae. These stressors are known to pose a threat to long-term survival of honey bees (Apis mellifera) and stability of the ecosystems. The research provides valuable insights into the adaptive mechanisms of honey bees in response to multiple stressors and developing effective conservation strategies. Further research can identify traits that promote honey bee survival in the face of future challenges from multiple stressors to maintain the overall stability of environment.

Overcoming the egress block of Plasmodium sporozoites expressing fluorescently tagged circumsporozoite protein.

Plasmodium sporozoites are the highly motile and invasive forms of the malaria parasite transmitted by mosquitoes. Sporozoites form within oocysts at the midgut wall of the mosquito, egress from oocysts and enter salivary glands prior to transmission. The GPI-anchored major surface protein, the circumsporozoite protein (CSP) is important for Plasmodium sporozoite formation, egress, migration and invasion. To visualize CSP, we previously generated full-length versions of CSP internally tagged with the green fluorescent protein, GFP. However, while these allowed for imaging of sporogony in oocysts, sporozoites failed to egress. Here, we explore different strategies to overcome this block in egress and obtain salivary gland resident sporozoites that express CSP-GFP. Replacing the N-terminal and repeat region with GFP did not allow sporozoite formation. Lowering expression of CSP-GFP at the endogenous locus allowed sporozoite formation but did not overcome egress block. Crossing of CSP-GFP expressing parasites that are blocked in egress with wild-type parasites yielded a small fraction of parasites that entered salivary glands and expressed various levels of CSP-GFP. Expressing CSP-GFP constructs from a silent chromosome region from promoters that are active only post salivary gland invasion yielded normal numbers of fluorescent salivary gland sporozoites, albeit with low levels of fluorescence. We also show that lowering CSP expression by 50% allowed egress from oocysts but not salivary gland entry. In conclusion, Plasmodium berghei parasites with normal CSP expression tolerate a certain level of CSP-GFP without disruption of oocyst egress and salivary gland invasion.

Searching for new molecules involved in Anopheles mosquitoes' response to Plasmodium infection.

Plasmodium parasites within mosquitoes are exposed to various physiological processes, such as blood meal digestion activity, the gonotrophic cycle, and host responses preventing the entry of parasites into the midgut wall. However, when in vitro-cultured ookinetes are injected into the hemocoel of mosquitoes, Plasmodium parasites are not affected by the vertebrate host's blood contents and do not pass through the midgut epithelial cells. This infection method might aid in identifying mosquito-derived factors affecting Plasmodium development within mosquitoes. This study investigated novel mosquito-derived molecules related to parasite development in Anopheles mosquitoes. We injected in vitro-cultured Plasmodium berghei (ANKA strain) ookinetes into female and male Anopheles stephensi (STE2 strain) mosquitoes and found that the oocyst number was significantly higher in males than in females, suggesting that male mosquitoes better support the development of parasites. Next, RNA-seq analysis was performed on the injected female and male mosquitoes to identify genes exhibiting changes in expression. Five genes with different expression patterns between sexes and greatest expression changes were identified as being potentially associated with Plasmodium infection. Two of the five genes also showed expression changes with infection by blood-feeding, indicating that these genes could affect the development of Plasmodium parasites in mosquitoes.

The essential role of clathrin-mediated endocytosis and early endosomes in the trafficking of begomoviruses through the primary salivary glands of their whitefly vectors.

Many plant viruses are transmitted by insect vectors in a circulative manner. For efficient transmission, the entry of the virus from vector hemolymph into the primary salivary gland (PSG) is a step of paramount importance. Yet, vector components mediating virus entry into PSG remain barely characterized. Here, we demonstrate the role of clathrin-mediated endocytosis and early endosomes in begomovirus entry into whitefly PSG. Our findings unravel the key components involved in begomovirus transport within the whitefly body and transmission by their whitefly vectors and provide novel clues for blocking begomovirus transmission.

Ame-miR-980-3p participates in autophagy-mediated midgut remodelling in Apis mellifera via targeting Atg2B.

Autophagy is a process that serves to degrade damaged proteins and organelles, thereby promoting cell homeostasis, differentiation, development and survival. Many miRNAs have been found to have regulatory roles in autophagy. In insects, it has been shown that autophagy is involved in hormone-regulated programmed cell death during metamorphic midgut remodelling. However, whether this is also true during the remodelling of the honey bee midgut is unclear. In the present study, we explored the relationship between autophagy and midgut remodelling and sought to identify miRNAs involved in this physiological process. We found that autophagy occurred during midgut remodelling and that the inhibition of autophagy resulted in midgut dysplasia in prepupae. Differentially expressed miRNAs enriched in the autophagy signalling pathway during midgut remodelling were identified by small RNA-seq. Ame-miR-980-3p, which targets the autophagy-related gene Atg2B, was screened out. Furthermore, abnormal expression of ame-miR-980-3p in the pupal stage led to the thinning of the midgut wall of newly emerged bees (NE). When ame-miR-980-3p expression was inhibited, the intestinal villi of NE bees became significantly shorter and sparse, and the lipid signal in the peritrophic matrix of Pb almost disappeared, indicating that the adult midgut was underdeveloped and the lipid absorption ability was weakened. Taken together, ame-miR-980-3p targeted Atg2B to participate in the regulation of midgut autophagy in the pupae, and the abnormal expression of ame-miR-980-3p would interfere with cell proliferation and death in the process of midgut remodelling, hinder the formation of adult midgut and eventually lead to adult midgut dysplasia and affect the lipid absorption function of the midgut in Apis mellifera.

Anatomical damage caused by Bacillus thuringiensis variety israelensis in yellow fever mosquito Aedes aegypti (L.) larvae revealed by micro-computed tomography

With micro-computed tomography techniques, using the single-distance phase-retrieval algorithm phase contrast, we reconstructed enhanced rendered images of soft tissues of Aedes aeqypti fourth instar larvae after Bti treatment. In contrast to previous publications based on conventional microscopy, either optical or electron microscopy, which were limited to partial studies, mostly in the form of histological sections, here we show for the first time the effects of Bti on the complete internal anatomy of an insect. Using 3D rendered images it was possible to study the effect of the bacterium in tissues and organs, not only in sections but also as a whole. We compared the anatomy of healthy larvae with the changes undergone in larvae after being exposed to Bti (for 30 min, 1 h and 6 h) and observed the progressive damage that Bti produce. Damage to the midgut epithelia was confirmed, with progressive swelling of the enterocytes, thickening epithelia, increase of the vacuolar spaces and finally cell lysis, producing openings in the midgut walls. Simultaneously, the larvae altered their motility, making it difficult for them to rise to the surface and position the respiratory siphon properly to break surface tension and breathe. Internally, osmotic shock phenomena were observed, resulting in a deformation of the cross-section shape, producing the appearance of a wide internal space between the cuticle and the internal structures and a progressive collapse of the tracheal trunks. Taken together, these results indicate the death of the larvae, not by starvation as a consequence of the destruction of the epithelia of the digestive tract as previously stated, but due to a synergic catastrophic multifactor process in addition to asphyxia due to a lack of adequate gas exchange.

Molecular identification of the chitinase genes in Aedes albopictus and essential roles of AaCht10 in pupal-adult transition

BackgroundAedes albopictus is an increasingly serious threat in public health due to it is vector of multiple arboviruses that cause devastating human diseases, as well as its widening distribution in recent years. Insecticide resistance is a serious problem worldwide that limits the efficacy of chemical control strategies against Ae. albopictus. Chitinase genes have been widely recognized as attractive targets for the development of effective and environmentally safe insect management measures.MethodsChitinase genes of Ae. albopictus were identified and characterized on the basis of bioinformatics search of the referenced genome. Gene characterizations and phylogenetic relationships of chitinase genes were investigated, and spatio-temporal expression pattern of each chitinase gene was evaluated using qRT-PCR. RNA interference (RNAi) was used to suppress the expression of AaCht10, and the roles of AaCht10 were verified based on phynotype observations, chitin content analysis and hematoxylin and eosin (H&E) stain of epidermis and midgut.ResultsAltogether, 14 chitinase-related genes (12 chitinase genes and 2 IDGFs) encoding 17 proteins were identified. Phylogenetic analysis showed that all these AaChts were classified into seven groups, and most of them were gathered into group IX. Only AaCht5-1, AaCht10 and AaCht18 contained both catalytic and chitin-binding domains. Different AaChts displayed development- and tissue-specific expression profiling. Suppression of the expression of AaCht10 resulted in abnormal molting, increased mortality, decreased chitin content and thinning epicuticle, procuticle and midgut wall of pupa.ConclusionsFindings of the present study will aid in determining the biological functions of AaChts and also contribute to using AaChts as potential target for mosquito management.Graphical

Occurrence of Gregarines (Protozoa: Apicomplexa) in the Neotropical Soldierless Ruptitermes spp. (Isoptera, Termitidae, Apicotermitinae)

This is the first record of gregarine infection in the worker midgut of the Neotropical termites Ruptitermes pitan and Ruptitermes reconditus. Gregarines have already been described in the gut and hemocoel in workers of other termite species, and although these protozoa are not able to kill these insects, they may affect their fitness. In the present study, gregarines were only observed in the gut lumen or linked to the midgut wall of termite workers. Histological sections of the worker midgut of Ruptitermes spp. showed three phases of the gregarine life cycle with the same individual worker displayed up to 4 gamonts and 2 trophozoites.

SYNERGISTIC EFFECT OF HONEY AND LEMONJUICE-ENRICHED MULBERRY DIETS ON THE DIGESTIVE METABOLISM OF THE SILKWORM, BOMBYX MOR

Synergistic impact of honey and lemon juice-enriched mulberry diets has been studied on the digestive metabolism of fth instar larval Bombyx mori. The study focused on the digestibility of proteins, carbohydrates, sucrose and cellulose by their digestive enzymes in the larval midgut wall and midgut lumen compartments and biomass accumulation in the gut wall. The honey and lemon juice-enriched diets showed positive impact on protease activity and protein digestion, but did not show discernable effect on the activity levels of α-amylase, sucrase and cellulase and the digestibility of carbohydrates, sucrose and cellulose. Nevertheless, they signicantly reinforced the digestive mass accumulation in gut wall cells in accordance with the Hutchinson's investment principle. Further, as evidenced by higher growth rates in the digestive parameters during rst ve days of the fth instar regime, the rate of biomass accumulation is ne-tuned by the timing of acquisition of critical larval body size determinants.

The Effect of Glucosinolates on the Growth and Development of Helicoverpa armigera Larvae and the Expression of Midgut Sulfatase Genes

The plant–pest interaction and its mechanisms are a novel research direction for pest control. They provide molecular targets for developing new pesticides and targeted control measures to control insect herbivores. Glucosinolate is a large family of secondary substances found in cruciferous plants that are harmful to herbivorous insects. Specialist herbivores have developed specific anti-defense genes and detoxifying mechanisms against glucosinolate from the host plant, but how generalist herbivores respond to glucosinolate at the molecular level is unknown. In this study, we investigated the effects of different glucosinolate concentrations on the growth and development of Helicoverpa armigera. Moreover, the expression of sulfatase genes (HaSulfs) was also checked following exposure to glucosinolate concentrations. The developmental duration of larvae and pre-pupa of H. armigera was significantly increased by 14.79–25.03% after feeding glucosinolate compared to the control. Quantitative Real-Time PCR (RT-qPCR) was carried out to analyze the expression of HaSulf family genes in the midgut of fifth instar larvae of H. armigera. The results showed that the upregulated expression patterns of HaSulf family genes were diversified after feeding at different concentrations. The expression level of HaSulf was detected with the HaSulf antibody. Only the glucosinolate-fed larvae had a visible target band and were mainly distributed in the midgut wall. Taken together, glucosinolate can significantly affect the growth and development of H. armigera larvae. It can induce the expression of HaSulf in the midgut of H. armigera at gene and protein levels. This study could be useful to understand the development of plant-derived insecticides resistance in H. armigera.

PSOP1, putative secreted ookinete protein 1, is localized to the micronemes of Plasmodium yoelii and P. berghei ookinetes.

Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Activated gametocytes in the mosquito midgut egress from erythrocytes followed by fertilization and zygote formation. Zygotes differentiate into motile invasive ookinetes, which penetrate the midgut epithelium before forming oocysts beneath the basal lamina. Ookinete development and traversal across the mosquito midgut wall are major bottlenecks in the parasite life cycle. In ookinetes, surface proteins and proteins stored in apical organelles have been shown to be involved in parasite-host interactions. A group of ookinete proteins that are predicted to have such functions are named PSOPs (putative secreted ookinete protein). PSOP1 is possibly involved in migration through the midgut wall, and here its subcellular localization was examined in ookinetes by immunoelectron microscopy. PSOP1 localizes to the micronemes of Plasmodium yoelii and Plasmodium berghei ookinetes, indicating that it is stored and possibly apically secreted during ookinete penetration through the mosquito midgut wall.

Dissecting Out the Molecular Mechanism of Insecticidal Activity of Ostreolysin A6/Pleurotolysin B Complexes on Western Corn Rootworm.

Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut.

Gut-Expressed Vitellogenin Facilitates the Movement of a Plant Virus across the Midgut Wall in Its Insect Vector.

ABSTRACTMany viral pathogens of global importance to plant and animal health are persistently transmitted by insect vectors. Midgut of insects forms the first major barrier that these viruses encounter during their entry into the vectors. However, the vector ligand(s) involved in the movement of plant viruses across the midgut barrier remains largely uncharacterized. Begomoviruses, many of which are disease agents of some major crops worldwide, are persistently transmitted by whiteflies (Bemisia tabaci). Here, in order to identify whitefly midgut proteins that interact with a devastating begomovirus, tomato yellow leaf curl virus (TYLCV), we performed midgut-specific TYLCV coat protein (CP) immunoprecipitation followed by high-throughput mass spectrometry proteomic analysis. We find that vitellogenin (Vg), a critical insect reproductive protein that has been considered to be synthesized by the fat body, is also synthesized by and interacts with TYLCV CP in the whitefly midgut. TYLCV appears to be internalized into midgut epithelial cells as a complex with Vg through endocytosis. Virus-containing vesicles then deliver the virus-Vg complexes to early endosomes for intracellular transport. Systematic silencing of Vg or midgut-specific immune blocking of Vg inhibited virus movement across the midgut wall and decreased viral acquisition and transmission by whitefly. Our findings show that a functional Vg protein is synthesized in the midgut of an insect and suggest a novel Vg mechanism that facilitates virus movement across the midgut barrier of its insect vector.IMPORTANCE An essential step in the life cycle of many viruses is transmission to a new host by insect vectors, and one critical step in the transmission of persistently transmitted viruses is overcoming the midgut barrier to enter vectors and complete their cycle. Most viruses enter vector midgut epithelial cells via specific interaction between viral structural proteins and vector cell surface receptor complexes. Tomato yellow leaf curl virus (TYLCV) is persistently transmitted by the whitefly Bemisia tabaci between host plants. Here, we find that TYLCV coat protein interacts with vitellogenin (Vg) in the whitefly midgut. This interaction is required for the movement of the virus crossing the midgut wall and thus facilitates viral acquisition and transmission by whitefly. This study reveals a novel mechanism of virus overcoming the insect midgut barrier and provides new insights into the function of Vg beyond serving as nutrition for developing embryos in insects.

Volatile DMNT directly protects plants against Plutella xylostella by disrupting the peritrophic matrix barrier in insect midgut.

Insect pests negatively affect crop quality and yield; identifying new methods to protect crops against insects therefore has important agricultural applications. Our analysis of transgenic Arabidopsis thaliana plants showed that overexpression of pentacyclic triterpene synthase 1, encoding the key biosynthetic enzyme for the natural plant product (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), led to a significant resistance against a major insect pest, Plutella xylostella. DMNT treatment severely damaged the peritrophic matrix (PM), a physical barrier isolating food and pathogens from the midgut wall cells. DMNT repressed the expression of PxMucin in midgut cells, and knocking down PxMucin resulted in PM rupture and P. xylostella death. A 16S RNA survey revealed that DMNT significantly disrupted midgut microbiota populations and that midgut microbes were essential for DMNT-induced killing. Therefore, we propose that the midgut microbiota assists DMNT in killing P. xylostella. These findings may provide a novel approach for plant protection against P. xylostella.

Characterization of the Temporal Pattern of Blood Protein Digestion in Rhodnius prolixus: First Description of Early and Late Gut Cathepsins.

Rhodnius prolixus is one important vector for the parasite Trypanosoma cruzi in Latin America, where Chagas disease is a significant health issue. Although R. prolixus is a model for investigations of vector–parasite interaction and transmission, not much has been done recently to further comprehend its protein digestion. In this work, gut proteolysis was characterized using new fluorogenic substrates, including optimum pH, inhibition profiles, and tissue and temporal expression patterns. Each protease possessed a particular tissue prevalence and activity cycle after feeding. Cathepsin L had a higher activity in the posterior midgut lumen, being characterized by a plateau of high activities during several days in the intermediate phase of digestion. Cathepsin D showed high activity levels in the tissue homogenates and in the luminal content of the posterior midgut, with a single peak 5 days after blood feeding. Aminopeptidases are highly associated with the midgut wall, where the highest activity is located. Assays with proteinaceous substrates as casein, hemoglobin, and serum albumin revealed different activity profiles, with some evidence of biphasic temporal proteolytic patterns. Cathepsin D genes are preferentially expressed in the anterior midgut, while cathepsin L genes are mainly located in the posterior portion of the midgut, with specific sets of genes being differently expressed in the initial, intermediate, or late phases of blood digestion.Significance StatementThis is the first description in a non-dipteran hematophagous species of a sequential protease secretion system based on midgut cathepsins instead of the most common insect digestive serine proteases (trypsins and chymotrypsins). The midgut of R. prolixus (Hemiptera) shows a different temporal expression of proteases in the initial, intermediate, and late stages of blood digestion. In this respect, a different timing in protease secretion may be an example of adaptative convergence in blood-sucking vectors from different orders. Expanding the knowledge about gut physiology in triatomine vectors may contribute to the development of new control strategies, aiming the blocking of parasite transmission.

A Hetero-Multimeric Chitinase-Containing Plasmodium falciparum and Plasmodium gallinaceum Ookinete-Secreted Protein Complex Involved in Mosquito Midgut Invasion.

Malaria parasites are transmitted by Anopheles mosquitoes. During its life cycle in the mosquito vector the Plasmodium ookinete escapes the proteolytic milieu of the post-blood meal midgut by traversing the midgut wall. This process requires penetration of the chitin-containing peritrophic matrix lining the midgut epithelium, which depends in part on ookinete-secreted chitinases. Plasmodium falciparum ookinetes have one chitinase (PfCHT1), whereas ookinetes of the avian-infecting parasite, P. gallinaceum, have two, a long and a short form, PgCHT1 and PgCHT2, respectively. Published data indicates that PgCHT2 forms a high molecular weight (HMW) reduction-sensitive complex; and one binding partner is the ookinete-produced von Willebrand A-domain-containing protein, WARP. Size exclusion chromatography data reported here show that P. gallinaceum PgCHT2 and its ortholog, P. falciparum PfCHT1 are covalently-linked components of a HMW chitinase-containing complex (> 1,300 kDa). Mass spectrometry of ookinete-secreted proteins isolated using a new chitin bead pull-down method identified chitinase-associated proteins in P. falciparum and P. gallinaceum ookinete-conditioned culture media. Mass spectrometry of this complex showed the presence of several micronemal proteins including von Willebrand factor A domain-related protein (WARP), ookinete surface enolase, and secreted ookinete adhesive protein (SOAP). To test the hypothesis that ookinete-produced PfCHT1 can form a high molecular homo-multimer or, alternatively, interacts with P. berghei ookinete-produced proteins to produce an HMW hetero-multimer, we created chimeric P. berghei parasites expressing PfCHT1 to replace PbCHT1, enabling the production of large numbers of PfCHT1-expressing ookinetes. We show that chimeric P. berghei ookinetes express monomeric PfCHT1, but a HMW complex containing PfCHT1 is not present. A better understanding of the chitinase-containing HMW complex may enhance development of next-generation vaccines or drugs that target malaria transmission stages.

A vector whitefly endocytic receptor facilitates the entry of begomoviruses into its midgut cells via binding to virion capsid proteins.

Many circulative plant viruses transmitted by insect vectors are devastating to agriculture worldwide. The midgut wall of vector insects represents a major barrier and at the same time the key gate a circulative plant virus must cross for productive transmission. However, how these viruses enter insect midgut cells remains poorly understood. Here, we identified an endocytic receptor complex for begomoviruses in the midgut cells of their whitefly vector. Our results show that two whitefly proteins, BtCUBN and BtAMN, compose a receptor complex BtCubam, for which BtCUBN contributes a viral-binding region and BtAMN contributes to membrane anchorage. Begomoviruses appear to be internalized together with BtCubam via its interaction with the 12–19 CUB domains of BtCUBN via clathrin-dependent endocytosis. Functional analysis indicates that interruption of BtCUBN and BtAMN lead to reduction of virus acquisition and transmission by whitefly. In contrast, CUBN-begomovirus interaction was not observed in two non-competent whitefly-begomovirus combinations. These observations suggest a major role of the specific endocytic receptor in facilitating viral entry into vector midgut cells.