Tandem Mass Tag-Based Quantitative Proteomics and Virulence Phenotype of Hemolymph-Treated Bacillus thuringiensis kurstaki Cells Reveal New Insights on Bacterial Pathogenesis in Insects

After ingestion by a susceptible insect and damaging its midgut epithelium, the bacterium Bacillus thuringiensis (Bt) reaches the insect blood (hemolymph), where it propagates despite the host’s antimicrobial defenses and induces insect death by acute septicemia. Although the hemolymph stage of the Bt toxic pathway is determinant for the infested insects’ fate, the response of Bt to hemolymph and the latter’s role in bacterial pathogenesis has been poorly explored.

Structure of the midgut epithelium in four diplopod species: histology, histochemistry and ultrastructure

The middle region of the digestive system of millipedes, the midgut, is responsible for all processes connected with digestion, but also takes part in homeostasis maintenance thanks to the ability to activate many mechanisms which neutralize changes occurring at different levels of the animal’s body. Numerous millipede species are treated as bioindicators of the natural environment and they are exposed to different stressors which originate from external environment. To obtain all data on the functioning of midgut of millipedes as the barrier against stressors, it is necessary to have a precise and general description of the midgut epithelium. Members from four millipede orders were selected for the studies: Polydesmus angustus (Polydesmida), Epibolus pulchripes (Spirobolida), Unciger transsilvanicus (Julida) and Glomeris tetrasticha (Glomerida). The structure and ultrastructure of their midgut epithelial cells (the digestive, secretory and regenerative cells) were documented using transmission electron microscopy and histochemical methods. The obtained results have been compared and discussed to previous ones, to present the general and structural organization of the midgut in Diplopoda. Our studies revealed that the ultrastructure of all cells which form the midgut epithelium in millipedes is general for all the species studied up to now and it resembles the cell ultrastructure observed in Chilopoda and Hexapoda, including the digestive, secretory and stem cells.

A malaria parasite phospholipid flippase safeguards midgut traversal of ookinetes for mosquito transmission

Mosquito midgut epithelium traversal is essential for malaria parasite transmission. Phospholipid flippases are eukaryotic type 4 P-type adenosine triphosphatases (P4-ATPases), which, in association with CDC50, translocate phospholipids across the membrane lipid bilayers. In this study, we investigated the function of a putative P4-ATPase, ATP7, from the rodent malaria parasite Plasmodium yoelii. Disruption of ATP7 blocks the parasite infection of mosquitoes. ATP7 is localized on the ookinete plasma membrane. While ATP7-depleted ookinetes are capable of invading the midgut, they are eliminated within the epithelial cells by a process independent from the mosquito complement-like immunity. ATP7 colocalizes and interacts with the flippase cofactor CDC50C. Depletion of CDC50C phenocopies ATP7 deficiency. ATP7-depleted ookinetes fail to uptake phosphatidylcholine across the plasma membrane. Ookinete microinjection into the mosquito hemocoel reverses the ATP7 deficiency phenotype. Our study identifies Plasmodium flippase as a mechanism of parasite survival in the midgut epithelium that is required for mosquito transmission.

Peritrophic matrix-degrading proteins are dispensable virulence factors in a virulent Melissococcus plutonius strain

European foulbrood (EFB) caused by Melissococcus plutonius is a major bacterial disease of honey bees. Strains of the causative agent exhibit genetic heterogeneity, and the degree of virulence varies among strains. In bee larvae orally infected with the highly virulent strains, ingested bacterial cells colonize the larval midgut and proliferate within the sac of the peritrophic matrix (PM), a barrier lining the midgut epithelium. However, the barrier is degraded during the course of infection, and M. plutonius cells eventually directly interact with the midgut epithelium. As M. plutonius possesses genes encoding putative PM-degrading proteins (enhancin, a chitin-binding domain-containing protein and endo-α-N-acetylgalactosaminidase), we constructed PM-degrading protein gene-knockout mutants from a highly virulent M. plutonius strain and investigated their role in the pathogenesis of EFB. In larvae infected with the triple-knockout mutant, which has no PM-degrading protein genes, M. plutonius that proliferated in the larval midguts was confined to the sac of the PM. However, the midgut epithelial cells degenerated over time, and the mutant killed approximately 70–80% of bee brood, suggesting that although the PM-degrading proteins are involved in the penetration of the PM by M. plutonius, they are not indispensable virulence factors in the highly virulent M. plutonius strain.

Infection of Aedes aegypti mosquitoes with midgut-attenuated Sindbis virus reduces, but does not eliminate, disseminated infection

Arboviruses are transmitted by specific vectors and the reasons for this specificity are not fully understood. One contributing factor is the existence of tissue barriers within the vector such as the midgut escape barrier. We used miRNA targeting of Sindbis virus (SINV) to study how replication in midgut cells contributes to overcoming this barrier in the mosquito Aedes aegypti. SINV constructs were designed to be attenuated specifically in midgut cells by inserting binding sites for midgut-specific miRNAs into either the 3′ untranslated region (MRE3′miRT) or the structural open reading frame (MRE-ORFmiRT) of the SINV genome. Both miRNA-targeted viruses replicated less efficiently than control viruses in the presence of these miRNAs. When mosquitoes were given infectious blood meals containing miRNA-targeted viruses, only around 20% (MRE3′miRT) or 40% (MRE-ORFmiRT) of mosquitoes developed disseminated infection. In contrast, dissemination occurred in almost all mosquitoes fed control viruses. Deep sequencing of virus populations from individual mosquitoes ruled out selection for mutations in the inserted target sequences as being the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to control virus in both the midgut and the carcass and there was no evidence of a threshold titer necessary for dissemination. Instead, it appeared that if infection was successfully established in the midgut, replication and dissemination were largely normal. Our results support the hypothesis that replication is an important factor in allowing SINV to overcome the midgut escape barrier, but hint that other factors are also likely involved. IMPORTANCE When a mosquito ingests an arbovirus during a blood meal, the arbovirus must escape from the midgut of the vector and infect the salivary glands in order to be transmitted to a new host. We used tissue-specific miRNA targeting to examine the requirement for Sindbis virus (SINV) to replicate in midgut epithelium in order to cause disseminated infection in the mosquito Aedes aegypti. Our results indicate that specifically reducing the ability of SINV to replicate in the mosquito midgut reduces its overall ability to establish infection in the mosquito, but if infection is established, replication and dissemination occur normally. These results are consistent with an importance for replication in the midgut epithelium in aiding arboviruses in crossing the midgut barrier.

Insight Into Cellular Uptake and Transcytosis of Peptide Nanoparticles in a Generalist Caterpillar.

Development of novel and specific insect pest management methods is critical for overcoming pesticide resistance and off-target effects. Gene silencing through consumption of double stranded (dsRNA) by insects shows promise in this area. Association of dsRNA with nanoparticles confers protection against nucleases, and can also promote translocation of dsRNA across the midgut epithelial cell membranes, and overall enhance gene knockdown effects. However, many mysteries of how nanoparticles and dsRNA are internalized by cells and subsequently transported across the midgut epithelium remain to be unraveled. In this article, we investigate the role of endocytosis and transcytosis in the uptake and transport of dsRNA and nanoparticles through midgut epithelium cells. Spodoptera frugiperda (Sf9) cells and branched amphiphilic peptide nano-capsules (BAPCs) were used as an experimental model. Additionally, analyses of reactive oxygen and nitrogen species (ROS/RNS) were performed to demonstrated that cell viability was minimally impacted by the BAPCs-dsRNA complex. Results suggests that clathrin-mediated endocytosis and macropinocytois are largely responsible for cellular uptake, and once within the midgut, transcytosis is involved in shuttling BAPCs from the lumen to the hemolymph. BAPCs were not found to be toxic to Sf9 cells or generate damaging reactive species once internalized. This opens up further possibilities for BAPCs as a new insect pest management method.

A malaria parasite phospholipid flippase safeguards midgut traversal of ookinetes for mosquito transmission

Mosquito midgut epithelium traversal is an essential component of transmission of malaria parasites. Phospholipid flippases are eukaryotic type IV ATPases (P4-ATPases), which in association with CDC50 cofactors, translocate phospholipids across lipid bilayers to maintain the membrane asymmetry. In this study, we investigated the function of a putative P4-ATPase, ATP7, from the rodent malaria parasite P. yoelii. Disruption of ATP7 results in block of parasite infection of mosquitoes. ATP7 is localized on the ookinete plasma membrane. While ATP7-depleted ookinetes are motile and capable of invading the midgut, they are quickly eliminated within the epithelial cells by a process that is independent from the mosquito complement-like immunity. ATP7 colocalizes and interacts with the flippase co-factor CDC50C. Importantly, depletion of CDC50C phenocopies ATP7 deficiency. ATP7-depleted ookinetes fail to translocate phosphatidylcholine (PC) across the plasma membrane, resulting in PC exposure at the ookinete surface. Lastly, ookinete microinjection into the mosquito hemocoel reverses the ATP7 deficiency phenotype. Our study identifies Plasmodium flippase as a novel mechanism of parasite survival in the midgut epithelium that is required for mosquito transmission.

Na+/H+-exchangers differentially contribute to midgut fluid sodium and proton concentration in the sea urchin larva

Regulation of ionic composition and pH is a requisite of all digestive systems in the animal kingdom. Larval stages of the marine superphylum ambulacraria, including echinoderms and hemichordates, were demonstrated to have highly alkaline conditions in their midgut with the underlying epithelial transport mechanisms being largely unknown.Using ion-selective microelectrodes, the present study demonstrated that pluteus larvae of the purple sea urchin have highly alkaline pH (pH ∼9) and low [Na+] ( ̴120 mM) in their midgut fluids, compared to the ionic composition of the surrounding sea water. We pharmacologically investigated the role of Na+/H+-exchangers in intracellular pH regulation and midgut proton and sodium maintenance using the NHE inhibitor 5-(n-ethyl-n-isopropyl)amiloride (EIPA). Basolateral EIPA application decreased midgut pH while luminal application, via micro-injections increased midgut [Na+], without affecting pH. Immunohistochemical analysis demonstrated a luminal localization of NHE-2 (SpSlc9a2) in the midgut epithelium. Specific knockdown of spslc9a2 using vivo morpholinos led to an increase in midgut [Na+] without affecting pH. Acute acidification experiments in combination with qPCR analysis and measurements of midgut pH and [Na+] identified two other NHE isoforms, Spslc9a7 and SpSlc9a8 that potentially contribute to the regulation of [Na+] and pH in midgut fluids.This work provides new insights to ion regulatory mechanisms in the midgut epithelium of sea urchin larvae. The involvement of NHEs in regulating pH and Na+ balance in midgut fluids shows conserved features to insect and vertebrate digestive systems and may contribute to the ability of sea urchin larvae to cope with changes in seawater pH.