Ectoperitrophic Space Research Articles

A novel and dual digestive symbiosis scales up the nutrition and immune system of the holobiont Rimicaris exoculata

BackgroundIn deep-sea hydrothermal vent areas, deprived of light, most animals rely on chemosynthetic symbionts for their nutrition. These symbionts may be located on their cuticle, inside modified organs, or in specialized cells. Nonetheless, many of these animals have an open and functional digestive tract. The vent shrimp Rimicaris exoculata is fueled mainly by its gill chamber symbionts, but also has a complete digestive system with symbionts. These are found in the shrimp foregut and midgut, but their roles remain unknown. We used genome-resolved metagenomics on separate foregut and midgut samples, taken from specimens living at three contrasted sites along the Mid-Atlantic Ridge (TAG, Rainbow, and Snake Pit) to reveal their genetic potential.ResultsWe reconstructed and studied 20 Metagenome-Assembled Genomes (MAGs), including novel lineages of Hepatoplasmataceae and Deferribacteres, abundant in the shrimp foregut and midgut, respectively. Although the former showed streamlined reduced genomes capable of using mostly broken-down complex molecules, Deferribacteres showed the ability to degrade complex polymers, synthesize vitamins, and encode numerous flagellar and chemotaxis genes for host-symbiont sensing. Both symbionts harbor a diverse set of immune system genes favoring holobiont defense. In addition, Deferribacteres were observed to particularly colonize the bacteria-free ectoperitrophic space, in direct contact with the host, elongating but not dividing despite possessing the complete genetic machinery necessary for this.ConclusionOverall, these data suggest that these digestive symbionts have key communication and defense roles, which contribute to the overall fitness of the Rimicaris holobiont.FJvM6igKFE6x_yqS3roQ38Video

A reverse vaccinology approach on transmembrane carbonic anhydrases from Plasmodium species as vaccine candidates for malaria prevention

BackgroundMalaria is a significant parasitic infection, and human infection is mediated by mosquito (Anopheles) biting and subsequent transmission of protozoa (Plasmodium) to the blood. Carbonic anhydrases (CAs) are known to be highly expressed in the midgut and ectoperitrophic space of Anopheles gambiae. Transmembrane CAs (tmCAs) in Plasmodium may be potential vaccine candidates for the control and prevention of malaria.MethodsIn this study, two groups of transmembrane CAs, including α-CAs and one group of η-CAs were analysed by immunoinformatics and computational biology methods, such as predictions on transmembrane localization of CAs from Plasmodium spp., affinity and stability of different HLA classes, antigenicity of tmCA peptides, epitope and proteasomal cleavage of Plasmodium tmCAs, accessibility of Plasmodium tmCAs MHC-ligands, allergenicity of Plasmodium tmCAs, disulfide-bond of Plasmodium tmCAs, B cell epitopes of Plasmodium tmCAs, and Cell type-specific expression of Plasmodium CAs.ResultsTwo groups of α-CAs and one group of η-CAs in Plasmodium spp. were identified to contain tmCA sequences, having high affinity towards MHCs, high stability, and strong antigenicity. All putative tmCAs were predicted to contain sequences for proteasomal cleavage in antigen presenting cells (APCs).ConclusionsThe predicted results revealed that tmCAs from Plasmodium spp. can be potential targets for vaccination against malaria.

Basic Biology of Trypanosoma brucei with Reference to the Development of Chemotherapies.

Trypanosoma brucei are protozoan parasites that cause the lethal human disease African sleeping sickness and the economically devastating disease of cattle, Nagana. African sleeping sickness, also known as Human African Trypanosomiasis (HAT), threatens 65 million people and animal trypanosomiasis makes large areas of farmland unusable. There is no vaccine and licensed therapies against the most severe, late-stage disease are toxic, impractical and ineffective. Trypanosomes are transmitted by tsetse flies, and HAT is therefore predominantly confined to the tsetse fly belt in sub-Saharan Africa. They are exclusively extracellular and they differentiate between at least seven developmental forms that are highly adapted to host and vector niches. In the mammalian (human) host they inhabit the blood, cerebrospinal fluid (late-stage disease), skin, and adipose fat. In the tsetse fly vector they travel from the tsetse midgut to the salivary glands via the ectoperitrophic space and proventriculus. Trypanosomes are evolutionarily divergent compared with most branches of eukaryotic life. Perhaps most famous for their extraordinary mechanisms of monoallelic gene expression and antigenic variation, they have also been investigated because much of their biology is either highly unconventional or extreme. Moreover, in addition to their importance as pathogens, many researchers have been attracted to the field because trypanosomes have some of the most advanced molecular genetic tools and database resources of any model system. The following will cover just some aspects of trypanosome biology and how its divergent biochemistry has been leveraged to develop drugs to treat African sleeping sickness. This is by no means intended to be a comprehensive survey of trypanosome features. Rather, I hope to present trypanosomes as one of the most fascinating and tractable systems to do discovery biology.

Trypanosoma brucei colonizes the tsetse gut via an immature peritrophic matrix in the proventriculus.

The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles1. Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space. Although unproven, it is generally accepted that trypanosomes reach the ectoperitrophic space by penetrating the peritrophic matrix in the anterior midgut2-4. Here, we revisited this event using fluorescence- and electron-microscopy methodologies. We show that trypanosomes penetrate the ectoperitrophic space in which the newly made peritrophic matrix is synthesized by the proventriculus. Our model describes how these proventriculus-colonizing parasites can either migrate to the ectoperitrophic space or become trapped within peritrophic matrix layers to form cyst-like bodies that are passively pushed along the gut as the matrix gets remodelled. Furthermore, early proventricular colonization seems to be promoted by factors in trypanosome-infected blood that cause higher salivary gland infections and potentially increase parasite transmission.

Histology and ultramorphology of the mesenteric sacs in the Neotropical termite Neocapritermes opacus (Blattaria, Termitidae, Termitinae)

Neocapritermes opacus is a Neotropical termite, which feeds on a variety of food sources, including highly decayed wood and humus. Similar to many Termitidae, this species contains a mixed segment in the intestine, an anatomic distinct region composed of both mesenteric and proctodeal tissues. In Neocapritermes spp., a pair of intestinal protrusions referred to as mesenteric sacs are associated with the mixed segment. In the present study, with the aid of histological and scanning microscopy techniques, we provide a detailed anatomical and histological analyses of the mesenteric sacs in N. opacus workers. The results showed that the paired sacs are contiguous with the mesenteric component of the mixed segment and open into the ectoperitrophic space. The wall of the sacs is an epithelium composed of two types of cells (regenerative and principal) and musculature. The regenerative cells are located at the epithelium base and organized in regenerative crypts, while the principal cells are columnar and enfold the lumen of the sacs. Brush border was observed in the principal cell apex, aligned closely to rod-like bacteria. Secretory activity was observed in the principal cells and vesicles cast from them toward the sacs lumen, probably result of an apocrine activity. The sac epithelium was of mesenteric affiliation but differed from the intestinal midgut. The proctodeal epithelium was markedly different. Although some studies have been interpreted the mesenteric sacs as Malpighian nodules or Malpighian sacs, our results support that these structures are morphologically distinct and that the occurrence of mesenteric sacs is a synapomorphy for Neocapritermes. The precise function of the mesenteric sacs in termite digestion remains unclear; therefore, further investigations should carefully consider their secretory activity and symbiont population, aiming to understand their development and maintenance among these Neotropical species.

Aspectos morfo-fisiológicos del tracto digestivo en larvas de IV estadio de Bradysia impatien (Diptera: Sciaridae)

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.

Absence of a microbiome in the midgut trunk of six representative Crustacea

AbstractIt has become a common expectation for an animal’s guts to be a site for bacterial symbiosis, which may play a role in influencing various aspects of physiology including digestion and immune responses. The presence of a microbiome in the digestive tracts of crustaceans has been demonstrated in the past using molecular techniques and has encouraged manipulations including probiotics to enhance growth rates in cultured species. We suggest, however, that bacteria are restricted to the lumen of the gut and separated from the epithelium by the peritrophic matrix, which prevents their establishment as a permanent component of the gut. The peritrophic matrix, found in most arthropods, is a chitinous sheet containing accessory molecules, and is continuously formed by delamination from the epithelium of the midgut such that the actual surface is constantly being cleaned. The lifted layer then surrounds ingested materials, including microbes, in an impenetrable wrapper compartmentalizing digestive and immune functions. The ectoperitrophic space, lying between the peritrophic matrix and the epithelium, was examined in six species representatives of six groups of crustaceans (Branchiopoda, Amphipoda, Copepoda, and Decapoda) for the presence of microbes using light and transmission electron microscopy as well as fluorescent probes for bacteria. The results suggest that a microbiome is lacking in several common groups of crustaceans.

Ultramorphology of the peritrophic matrix in bees (Hymenoptera: Apidae)

The midgut is a region of the alimentary canal of bees with the lumen lined by a peritrophic matrix (PM) that is composed of chitin and proteins, secreted in response to feeding stimulus. The PM in adult bees has been described only under histological aspects. The objective of this work was to describe the PM in different bee species with a scanning electron microscope. The PMs have different features among the species analyzed. In the stingless bee Melipona quadrifasciata (Meliponini), the PM has two layers and the surface associated with the ectoperitrophic space has a hexagonal sculpture, whereas the endoperitrophic surface shows some wrinkles. In the honey bee Apis mellifera (Apini), and in the solitary bees Centris fuscata (Centridini) and Epicharis flava (Centridini) the PM has a laminate aspect with thin and irregular layers with dense material covering their surface, likely found for non Apidae bees Megachile rotundata (Megachilidae) and Oxaea flavescens (Andrenidae). These data suggest that the PM varies according to the bee species, which may be due to phylogeny other than feeding habits.

Flagellar cAMP signaling controls trypanosome progression through host tissues

The unicellular parasite Trypanosoma brucei is transmitted between mammals by tsetse flies. Following the discovery that flagellar phosphodiesterase PDEB1 is required for trypanosomes to move in response to signals in vitro (social motility), we investigated its role in tsetse flies. Here we show that PDEB1 knockout parasites exhibit subtle changes in movement, reminiscent of bacterial chemotaxis mutants. Infecting flies with the knockout, followed by live confocal microscopy of fluorescent parasites within dual-labelled insect tissues, shows that PDEB1 is important for traversal of the peritrophic matrix, which separates the midgut lumen from the ectoperitrophic space. Without PDEB1, parasites are trapped in the lumen and cannot progress through the cycle. This demonstrates that the peritrophic matrix is a barrier that must be actively overcome and that the parasite’s flagellar cAMP signaling pathway facilitates this. Migration may depend on perception of chemotactic cues, which could stem from co-infecting parasites and/or the insect host.

The physiological differentiation along the midgut of Bombyx mori - inspirations from proteomics and gene expression patterns of the secreted proteins in the ectoperitrophic space.

The ectoperitrophic space (EcPS) between the insect midgut epithelial cells and the peritrophic matrix is an unexplored, clean resource for concentrated proteins secreted by the midgut epithelial cells, which offers an ideal opportunity to uncover the midgut functions. In this study, we used Bombyx mori as a model organism and performed comparative proteomic analyses of the secreted proteins in the EcPS at the feeding and wandering stages. A total of 372 proteins were identified from both stages and 70 proteins were predicted to be secreted. Amongst these proteins, 17 secreted digestive proteins were identified and their temporal and spatial transcriptional expression patterns demonstrated that all these proteins were up-regulated at the feeding stage and differentially expressed in different parts of the midgut. Proteins with nutrient reservoir activity and defence activity were found to be up-regulated at the wandering stage. This work is the first to show the presence of digestive enzymes in the EcPS of the insect midgut using a proteomic approach, which provides evidence that suggests a physiological functional differentiation of the insect midgut. It is very clear that the EcPS undergoes dynamic changes in its composition of proteins in response to the changing needs of the insect at different developmental stages.

A chitin-binding lectin from Moringa oleifera seeds (WSMoL) impairs the digestive physiology of the Mediterranean flour larvae, Anagasta kuehniella

Biotechnological techniques allow the investigation of alternatives to outdated chemical insecticides for crop protection; some investigations have focused on the identification of molecules tailored from nature for this purpose. We, herein, describe the negative effects of water-soluble lectin from Moringa oleifera seeds (WSMoL) on Anagasta kuehniella development. The chitin-binding lectin, WSMoL, impaired the larval weight gain by 50% and affected the activity of the pest's major digestive enzymes. The commitment of the digestive process became evident after controlled digestion studies, where the capacity of protein digestion was compromised by >90%. Upon acute exposure, the lectin was not resistant to digestion; however, chronic ingestion of WSMoL was able to reverse this feature. Thus, we show that resistance to digestion may not be a prerequisite for a lectin's ability to exert negative effects on larval physiology. The mechanism of action of WSMoL involves binding to chitin with possible disruption to the peritrophic membrane, causing disorder between the endo- and ectoperitrophic spaces. Additionally, results suggest that WSMoL may trigger apoptosis in gut cells, leading to the lower enzymatic activity observed in WSMoL-fed larvae. Although assays employing an artificial diet did not demonstrate effects of WSMoL on A. kuehniella mortality, this lectin may hold potential for exerting insecticide effects on other pest insects, as well for use in other experimental approaches, such as WSMoL-expressing plants. Moreover, the use of WSMoL with other biotechnological tools, such as ‘pyramid’ crops, may represent a strategy for delaying the evolution of pest resistance to transgenic crops, since its multiple site targets could act in synergism with other insecticide compounds.

A Glycosylation Mutant of Trypanosoma brucei Links Social Motility Defects In Vitro to Impaired Colonization of Tsetse Flies In Vivo.

Transmission of African trypanosomes by tsetse flies requires that the parasites migrate out of the midgut lumen and colonize the ectoperitrophic space. Early procyclic culture forms correspond to trypanosomes in the lumen; on agarose plates they exhibit social motility, migrating en masse as radial projections from an inoculation site. We show that an Rft1(-/-) mutant needs to reach a greater threshold number before migration begins, and that it forms fewer projections than its wild-type parent. The mutant is also up to 4 times less efficient at establishing midgut infections. Ectopic expression of Rft1 rescues social motility defects and restores the ability to colonize the fly. These results are consistent with social motility reflecting movement to the ectoperitrophic space, implicate N-glycans in the signaling cascades for migration in vivo and in vitro, and provide the first evidence that parasite-parasite interactions determine the success of transmission by the insect host.

Effects of proteinase inhibitor from Adenanthera pavonina seeds on short- and long term larval development of Aedes aegypti.

Currently, one of the major global public health concerns is related to the transmission of dengue/yellow fever virus by the vector Aedes aegypti. The most abundant digestive enzymes in Ae. aegypti midgut larvae are trypsin and chymotrypsin. Since protease inhibitors have the capacity to bind to and inhibit the action of insect digestive proteinases, we investigated the short- and long-term effects of Adenanthera pavonina seed proteinase inhibitor (ApTI) on Ae. aegypti larvae, as well as a possible mechanism of adaptation. ApTI had a significant effect on Ae. aegypti larvae exposed to a non-lethal concentration of ApTI during short- and long-duration assays, decreasing survival, weight and proteinase activities of midgut extracts of larvae. The zymographic profile of ApTI demonstrated seven bands; three bands apparently have trypsin-like activity. Moreover, the peritrophic membrane was not disrupted. The enzymes of ApTI-fed larvae were found to be sensitive to ApTI and to have a normal feedback mechanism; also, the larval digestive enzymes were not able to degrade the inhibitor. In addition, ApTI delayed larval development time. Histological studies demonstrated a degeneration of the microvilli of the posterior midgut region epithelium cells, hypertrophy of the gastric caeca cells and an augmented ectoperitrophic space in larvae. Moreover, Ae. aegypti larvae were incapable of overcoming the negative effects of ApTI, indicating that this inhibitor might be used as a promising agent against Ae. aegypti. In addition, molecular modeling and molecular docking studies were also performed in order to construct three-dimensional theoretical models for ApTI, trypsin and chymotrypsin from Ae. aegypti, as well as to predict the possible interactions and affinity values for the complexes ApTI/trypsin and ApTI/chymotrypsin. In this context, this study broadens the base of our understanding about the modes of action of proteinase inhibitors in insects, as well as the way insects adapt to them.

Social Motility of African Trypanosomes Is a Property of a Distinct Life-Cycle Stage That Occurs Early in Tsetse Fly Transmission

The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen—designated as early procyclic forms—express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4–7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.

Enhancement of the Musca domestica hytrosavirus infection with orally delivered reducing agents

House flies (Musca domestica L.) throughout the world are infected with the salivary gland hypertrophy virus MdSGHV (Hytrosaviridae). Although the primary route of infection is thought to be via ingestion, flies that are old enough to feed normally are resistant to infection per os, suggesting that the peritrophic matrix (PM) is a barrier to virus transmission. Histological examination of the peritrophic matrix of healthy flies revealed a multilaminate structure produced by midgut cells located near the proventriculus. SEM revealed the PM to form a confluent sheet surrounding the food bolus with pores/openings less than 10nm in diameter. TEM revealed the PM to be multilayered, varying in width from 350 to 900nm, and generally thinner in male than in female flies. When flies were fed on the reducing agents dithiothetriol (DTT) or tris (2-caboxyethyl)phosphine hydrochloride (TCEP) for 48h before per os exposure to the virus, infection rates increased 10- to 20-fold compared with flies that did not receive the reducing agent treatments. PM’s from flies treated with DTT and TCEP displayed varying degrees of disruption, particularly in the outer layer, and lacked the electron-dense inner layer facing the ectoperitrophic space. Both drugs were somewhat toxic to the flies, resulting in>40% mortality at doses greater than 10mM (DTT) or 5mM (TCEP). DTT increased male fly susceptibility (55.1% infected) more than that of females (7.8%), whereas TCEP increased susceptibility of females (42.9%) more than that of males (26.2%). The cause for the sex differences in response to oral exposure the reducing agents is unclear. Exposing flies to food treated with virus and the reducing agents at the same time, rather than pretreating flies with the drugs, had no effect on susceptibility to the virus. Presumably, the reducing agent disrupted the enveloped virus and acted as a viricidal agent. In summary, it is proposed that the reducing agents influence integrity of the PM barrier and increase the susceptibility of flies to infection by MdSGHV.

A Lepidopteran-Specific Gene Family Encoding Valine-Rich Midgut Proteins

Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM), an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps), which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran digestive tract facing particular immune challenges.

Ookinete destruction within the mosquito midgut lumen explains Anopheles albimanus refractoriness to Plasmodium falciparum (3D7A) oocyst infection

Previous studies have shown that the central American mosquito vector, Anopheles albimanus, is generally refractory to oocyst infection with allopatric isolates of the human malaria parasite Plasmodium falciparum. However, the reasons for the refractoriness of A. albimanus to infection with such isolates of P. falciparum are unknown. In the current study, we investigated the infectivity of the P. falciparum clone 3D7A to laboratory-reared A. albimanus and another natural vector of human malaria, Anopheles stephensi. Plasmodium falciparum gametocytes grown in vitro were simultaneously fed to both mosquito species and the progress of malaria infection compared. In 22 independent paired experimental feeds, no mature oocysts were observed on the midguts of A. albimanus 10days after bloodfeeding. In contrast, high levels of oocyst infection were found on the midguts of simultaneously fed A. stephensi. Direct immunofluorescence microscopy and light microscopical examination of Giemsa-stained histological sections were used to identify when the P. falciparum clone 3D7A failed to establish mature oocyst infections in A. albimanus. Similar densities of macrogametes/zygotes, and immature retort-form and mature ookinetes were found within the bloodmeals of both mosquito species. However, in A. albimanus, ookinetes were seldom associated with the peritrophic matrix, and were neither observed in the ectoperitrophic space nor the midgut epithelium. In contrast, ookinetes were frequently observed in these midgut compartments in A. stephensi. Additionally, young oocysts were observed on the midguts of A. stephensi but not A. albimanus 2days after bloodfeeding. Vital staining of the immature retort-form and mature ookinetes found within the luminal bloodmeal, demonstrated that a significantly greater proportion of these malaria parasite stages were non-viable in A. albimanus compared with A. stephensi. Overall, our observations indicate that ookinetes of the P. falciparum clone 3D7A are destroyed within the bloodmeal of A. albimanus and that the midgut lumen, rather than the midgut epithelium, is the site of mosquito refractoriness in this particular malaria parasite-mosquito vector combination.

The development of Psychodiella sergenti (Apicomplexa: Eugregarinorida) in Phlebotomus sergenti (Diptera: Psychodidae).

SUMMARYPsychodiella sergenti is a recently described specific pathogen of the sand fly Phlebotomus sergenti, the main vector of Leishmania tropica. The aim of this study was to examine the life cycle of Ps. sergenti in various developmental stages of the sand fly host. The microscopical methods used include scanning electron microscopy, transmission electron microscopy and light microscopy of native preparations and histological sections stained with periodic acid-Schiff reaction. Psychodiella sergenti oocysts were observed on the chorion of sand fly eggs. In 1st instar larvae, sporozoites were located in the ectoperitrophic space of the intestine. No intracellular stages were found. In 4th instar larvae, Ps. sergenti was mostly located in the ectoperitrophic space of the intestine of the larvae before defecation and in the intestinal lumen of the larvae after defecation. In adults, the parasite was recorded in the body cavity, where the sexual development was triggered by a bloodmeal intake. Psychodiella sergenti has several unique features. It develops sexually exclusively in sand fly females that took a bloodmeal, and its sporozoites bear a distinctive conoid (about 700 nm long), which is more than 4 times longer than conoids of the mosquito gregarines.

The role of peritrophic membrane in the resistance of Anticarsia gemmatalis larvae (Lepidoptera: Noctuidae) during the infection by its nucleopolyhedrovirus ( AgMNPV)

The aim of this study was to analyze morphologically the peritrophic membrane (PM) of Anticarsia gemmatalis larvae resistant (RL) and non-resistant (susceptible) (SL) to the A. gemmatalis multicapsid nucleopolyhedrovirus ( AgMNPV), in the presence of viral infection. Also, in this investigation the results between SL and RL were compared to improve the understanding of the resistance mechanisms to the virus. The PM of SL of A. gemmatalis was less efficient as a barrier against the viral infection since it was found to be more fragile than the PM of RL. The lower chitin content as seen from weaker fluorescent staining in SL as well as the abundance of non-solubilized vesicular materials in the ectoperitrophic space, would cause the malformation of this membrane, facilitating the passage of the virus toward the epithelium of the midgut. On the other hand, in RL, the intensity of WGA (wheat germ agglutinin)-conjugated FITC (fluorescein) reaction of the PM was greater than in SL, making this insect more resistant to infection. We can conclude that the effectiveness of the PM in protecting against pathogens is dependent on the integrity of the epithelial cells of the midgut and of the structural preservation of the PM, being directly implicated in the resistance of A. gemmatalis larvae to AgMNPV.

A viral chitinase enhances oral activity of TMOF

In this study we investigate the combined effect on Heliothis virescens (Lepidoptera, Noctuidae) larvae of Aedes aegypti-Trypsin Modulating Oostatic Factor ( Aea-TMOF), a peptide that inhibits trypsin synthesis by the gut, impairing insect digestive function, and Autographa californica nucleopolyhedrovirus Chitinase A (AcMNPV ChiA), an enzyme that is able to alter the permeability of the peritrophic membrane (PM). Aea-TMOF and AcMNPV ChiA were provided to the larvae by administering transgenic tobacco plants, co-expressing both molecules. Experimental larvae feeding on these plants, compared to those alimented on plants expressing only one of the two molecules considered, showed significantly stronger negative effects on growth rate, developmental time and mortality. The impact of AcMNPV ChiA on the PM of H. virescens larvae, measured as increased permeability to molecules, was evident after five days of feeding on transgenic plants expressing ChiA. This result was confirmed by in vitro treatment of PM with recombinant ChiA, extracted from the transgenic plants used for the feeding experiments. Collectively, these data indicate the occurrence of a positive interaction between the two transgenes concurrently expressed in the same plant. The hydrolytic activity of ChiA on the PM of tobacco budworm larvae enhances the permeation of TMOF molecules to the ectoperitrophic space, and its subsequent absorption. The permeation through the paracellular route of Aea-TMOF resulted in a spotted accumulation on the basolateral domain of enterocytes, which suggests the occurrence of a receptor on the gut side facing the haemocoel. The binding of the peptide, permeating at increased rates due to the ChiA activity, is considered responsible for the enhanced insecticide activity of the transgenic plants expressing both molecules. These data corroborate the idea that ChiA can be effectively used as gut permeation enhancer in oral delivery strategies of bioinsecticides targeting haemocoelic receptors.