Midgut Cells Research Articles

Characteristics of channel pores formed by Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

Cry4Aa toxin produced by Bacillus thuringiensis subsp. israelensis exhibits specific toxicity to larvae of medically important mosquito genera. In the present study, we analyzed the characteristics of channel pores formed by recombinant Cry4Aa in a solvent-free planar lipid bilayer. Stable channel currents were observed in electrophysiologic measurements, and the single-channel conductance was 187 ± 10 pS in symmetrical buffer containing 150 mM KCl. The channel pores formed by Cry4Aa were cation-selective, with an estimated PK/PCl permeability ratio of 4.9. In addition, Cry4Aa channel pores exhibited apparent cation preference in the order Na+ > K+, Na+ > Ca2+, and K+ > Ca2+. Although the effect was limited, the cation preference of Cry4Aa channel pores seemed to be correlated with toxicity. Culex pipiens mosquito larvae reared in NaCl solution exhibited greater sensitivity to Cry4Aa, particularly early period after exposure. The presence of cations that preferentially translocate through Cry4Aa channel pores may facilitate excessive influx of water into the midgut cells, leading to colloid-osmotic lysis. Whereas CaCl2 had some effect on the mosquito-larvicidal activity of Cry4Aa, KCl had no effect. The effect of some cations may be mitigated by the variety of ion channels present on the midgut cell membrane.

Study supplementation of astaxanthin in high‐fat diet on growth performance, antioxidant ability, anti‐inflammation, non‐specific immunity and intestinal structure of juvenile Trachinotus ovatus

A 6-week feeding experiment was conducted to evaluate whether dietary supplementation of astaxanthin could mitigate negative effects of high-fat diet on growth performance, antioxidant capacity, anti-inflammation, non-specific immunity and intestinal structure of Trachinotus ovatus. Three isonitrogenous diets were formulated supplementing with or without high-fat or astaxanthin (lipid positive (LP): 130 g/kg lipid, lipid negative (LN): 210 g/kg lipid, lipid negative astaxanthin (LNA): 210 g/kg lipid with 0.5 g/kg astaxanthin). Results showed that the growth performance (weight gain (WG) and specific growth rate (SGR)) of T. ovatus in the LN group were significantly lower than that of growth performance in the LP group (p < .05), while growth performance data in the LNA group were significantly higher than those data in the LN group (p < .05) and without statistical difference with the LP group (p > .05). There were significantly lower levels of antioxidant contents (malondialdehyde (MDA) and Glutathione (GSH)) and transcriptional level of antioxidant-related gene (Nrf2, keap1, Mn-SOD, GSH-PX, HO-1 and CAT) in the LNA group when compared to the LN group (p < .05), while no statistical differences were detected between the LNA and LP groups (p > .05). NF-κB signal pathway key gene (p65) and non-specific immunity-related genes (IRF3, IRF7 and MyD88) of T. ovatus were significantly lower in the LNA and LP groups when compared to the LN group (p < .05). Fewer hepatic lipid droplets and more goblet cells of mid-gut were found in T. ovatus from the LNA group than those from the LN group. These results indicate that a diet supplementation of 0.5 g/kg astaxanthin mitigates detrimental effects of T. ovatus growth performance caused by high-fat diet by decreasing hepatic lipid accumulation and improving antioxidant, anti-inflammation, non-specific immunity and intestine barrier capacities.

Assessing the Impact of a Viral Infection on the Expression of Transposable Elements in the Cabbage Looper Moth (Trichoplusia ni).

Most studies of stress-induced transposable element (TE) expression have so far focused on abiotic sources of stress. Here, we analyzed the impact of an infection by the AcMNPV baculovirus on TE expression in a cell line (Tnms42) and midgut tissues of the cabbage looper moth (Trichoplusia ni). We find that a large fraction of TE families (576/636 in Tnms42 cells and 503/612 in midgut) is lowly expressed or not expressed at all [≤ 4 transcripts per million (TPM)] in the uninfected condition (median TPM of 0.37 in Tnms42 and 0.46 in midgut cells). In the infected condition, a total of 62 and 187 TE families were differentially expressed (DE) in midgut and Tnms42 cells, respectively, with more up- (46) than downregulated (16) TE families in the former and as many up- (91) as downregulated (96) TE families in the latter. Expression log2 fold changes of DE TE families varied from −4.95 to 9.11 in Tnms42 cells and from −4.28 to 7.66 in midgut. Large variations in expression profiles of DE TEs were observed depending on the type of cells and on time after infection. Overall, the impact of AcMNPV on TE expression in T. ni is moderate but potentially sufficient to affect TE activity and genome architecture. Interestingly, one host-derived TE integrated into AcMNPV genomes is highly expressed in infected Tnms42 cells. This result shows that virus-borne TEs can be expressed, further suggesting that they may be able to transpose and that viruses may act as vectors of horizontal transfer of TEs in insects.

Spatiotemporal organization of enteroendocrine peptide expression in Drosophila

The digestion of food and absorption of nutrients occurs in the gut. The nutritional value of food and its nutrients is detected by enteroendocrine cells, and peptide hormones produced by the enteroendocrine cells are thought to be involved in metabolic homeostasis, but the specific mechanisms are still elusive. The enteroendocrine cells are scattered over the entire gastrointestinal tract and can be classified according to the hormones they produce. We followed the changes in combinatorial expression of regulatory peptides in the enteroendocrine cells during metamorphosis from the larva to the adult fruit fly, and re-confirmed the diverse composition of enteroendocrine cell populations. Drosophila enteroendocrine cells appear to differentially regulate peptide expression spatially and temporally depending on midgut region and developmental stage. In the late pupa, Notch activity is known to determine which peptides are expressed in mature enteroendocrine cells of the posterior midgut, and we found that the loss of Notch activity in the anterior midgut results in classes of enteroendocrine cells distinct from the posterior midgut. These results suggest that enteroendocrine cells that populate the fly midgut can differentiate into distinct subtypes that express different combinations of peptides, which likely leads to functional variety depending on specific needs.

Design, Synthesis, and Study of the Insecticidal Activity of Novel Steroidal 1,3,4-Oxadiazoles.

A series of novel steroidal derivatives with a substituted 1,3,4-oxadiazole structure was designed and synthesized, and the target compounds were evaluated for their insecticidal activity against five aphid species. Most of the tested compounds exhibited potent insecticidal activity against Eriosoma lanigerum (Hausmann), Myzus persicae, and Aphis citricola. Compounds 20g and 24g displayed the highest activity against E. lanigerum, showing LC50 values of 27.6 and 30.4 μg/mL, respectively. Ultrastructural changes in the midgut cells of E. lanigerum were detected by transmission electron microscopy, indicating that these steroidal oxazole derivatives might exert their insecticidal activity by destroying the mitochondria and nuclear membranes in insect midgut cells. Furthermore, a field trial showed that compound 20g exhibited effects similar to those of the positive controls chlorpyrifos and thiamethoxam against E. lanigerum, reaching a control rate of 89.5% at a dose of 200 μg/mL after 21 days. We also investigated the hydrolysis and metabolism of the target compounds in E. lanigerum by assaying the activities of three insecticide-detoxifying enzymes. Compound 20g at 50 μg/mL exhibited inhibitory action on carboxylesterase similar to the known inhibitor triphenyl phosphate. The above results demonstrate the potential of these steroidal oxazole derivatives to be developed as novel pesticides.

Solanum nigrum Extract and Solasonine Affected Hemolymph Metabolites and Ultrastructure of the Fat Body and the Midgut in Galleria mellonella.

Glycoalkaloids, secondary metabolites abundant in plants belonging to the Solanaceae family, may affect the physiology of insect pests. This paper presents original results dealing with the influence of a crude extract obtained from Solanum nigrum unripe berries and its main constituent, solasonine, on the physiology of Galleria mellonella (Lepidoptera) that can be used as an alternative bioinsecticide. G. mellonella IV instar larvae were treated with S. nigrum extract and solasonine at different concentrations. The effects of extract and solasonine were evaluated analyzing changes in carbohydrate and amino acid composition in hemolymph by RP-HPLC and in the ultrastructure of the fat body cells by TEM. Both extract and solasonine changed the level of hemolymph metabolites and the ultrastructure of the fat body and the midgut cells. In particular, the extract increased the erythritol level in the hemolymph compared to control, enlarged the intracellular space in fat body cells, and decreased cytoplasm and lipid droplets electron density. The solasonine, tested with three concentrations, caused the decrease of cytoplasm electron density in both fat body and midgut cells. Obtained results highlighted the disturbance of the midgut and the fat body due to glycoalkaloids and the potential role of hemolymph ingredients in its detoxification. These findings suggest a possible application of glycoalkaloids as a natural insecticide in the pest control of G. mellonella larvae.

An image analysis method for regionally defined cellular phenotyping of the Drosophila midgut

SummaryThe intestine is divided into functionally distinct regions along the anteroposterior (A/P) axis. How the regional identity influences the function of intestinal stem cells (ISCs) and their offspring remain largely unresolved. We introduce an imaging-based method, “Linear Analysis of Midgut” (LAM), which allows quantitative, regionally defined cellular phenotyping of the whole Drosophila midgut. LAM transforms image-derived cellular data from three-dimensional midguts into a linearized representation, binning it into segments along the A/P axis. Through automated multivariate determination of regional borders, LAM allows mapping and comparison of cellular features and frequencies with subregional resolution. Through the use of LAM, we quantify the distributions of ISCs, enteroblasts, and enteroendocrine cells in a steady-state midgut, and reveal unprecedented regional heterogeneity in the ISC response to a Drosophila model of colitis. Altogether, LAM is a powerful tool for organ-wide quantitative analysis of the regional heterogeneity of midgut cells.

Rice black-streaked dwarf virus P10 promotes phosphorylation of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) to induce autophagy in Laodelphax striatellus

ABSTRACT Macroautophagy/autophagy is an important innate and adaptive immune response that can clear microbial pathogens through guiding their degradation. Virus infection in animals and plants is also known to induce autophagy. However, how virus infection induces autophagy is largely unknown. Here, we provide evidence that the early phase of rice black-streaked dwarf virus (RBSDV) infection in Laodelphax striatellus can also induce autophagy, leading to suppression of RBSDV invasion and accumulation. We have determined that the main capsid protein of RBSDV (P10) is the inducer of autophagy. RBSDV P10 can specifically interact with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), both in vitro and in vivo. Silencing of GAPDH in L. striatellus could significantly reduce the activity of autophagy induced by RBSDV infection. Furthermore, our results also showed that both RBSDV infection and RBSDV P10 alone can promote phosphorylation of AMP-activated protein kinase (AMPK), resulting in GAPDH phosphorylation and relocation of GAPDH from the cytoplasm into the nucleus in midgut cells of L. striatellus or Sf9 insect cells. Once inside the nucleus, phosphorylated GAPDH can activate autophagy to suppress virus infection. Together, these data illuminate the mechanism by which RBSDV induces autophagy in L. striatellus, and indicate that the autophagy pathway in an insect vector participates in the anti-RBSDV innate immune response. Abbreviations3-MA: 3-methyladenine; AMPK: AMP-activated protein kinase; ATG: autophagy-related; co-IP: co-immunoprecipitation; DAPI: 4ʹ,6-diamidino-2-phenylindole; dpf: days post-feeding; dsRNA: double-stranded RNA; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione-S-transferase; RBSDV: Rice black-streaked dwarf virus; TEM: transmission electron microscope.

RNA N6 -methyladenosine modification suppresses replication of rice black streaked dwarf virus and is associated with virus persistence in its insect vector.

N6 methylation of adenosine (m6A) was recently discovered to play a role in regulating the life cycle of various viruses by modifying viral and host RNAs. However, different studies on m6A effects on the same or different viruses have revealed contradictory roles for m6A in the viral life cycle. In this study, we sought to define the role of m6A on infection by rice black streaked dwarf virus (RBSDV), a double‐stranded RNA virus, of its vector small brown planthopper (SBPH). Infection by RBSDV decreased the level of m6A in midgut cells of SBPHs. We then cloned two genes (LsMETTL3 and LsMETTL14) that encode m6A RNA methyltransferase in SBPHs. After interference with expression of the two genes, the titre of RBSDV in the midgut cells of SBPHs increased significantly, suggesting that m6A levels were negatively correlated with virus replication. More importantly, our results revealed that m6A modification might be the epigenetic mechanism that regulates RBSDV replication in its insect vector and maintains a certain virus threshold required for persistent transmission.

Characterizing the Xenoma of Vairimorpha necatrix Provides Insights Into the Most Efficient Mode of Microsporidian Proliferation.

Microsporidia are a group of obligated intracellular parasites that can infect nearly all vertebrates and invertebrates, including humans and economic animals. Microsporidian Vairimorpha necatrix is a natural pathogen of multiple insects and can massively proliferate by making tumor-like xenoma in host tissue. However, little is known about the subcellular structures of this xenoma and the proliferation features of the pathogens inside. Here, we characterized the V. necatrix xenoma produced in muscle cells of silkworm midgut. In result, the whitish xenoma was initially observed on the 12th day post infection on the outer surface of the midgut and later became larger and numerous. The observation by scanning electronic microscopy showed that the xenoma is mostly elliptical and spindle with dense pathogen-containing protrusions and spores on the surface, which were likely shedding off the xenoma through exocytosis and could be an infection source of other tissues. Demonstrated with transmission electron microscopy and fluorescent staining, the xenoma was enveloped by a monolayer membrane, and full of vesicle structures, mitochondria, and endoplasmic reticulum around parasites in development, suggesting that high level of energy and nutrients were produced to support the massive proliferation of the parasites. Multiple hypertrophic nuclei were found in one single xenoma, indicating that the cyst was probably formed by fusion of multiple muscle cells. Observed by fluorescence in situ hybridization, pathogens in the xenoma were in merongony, sporogony, and octosporogony, and mature stages. And mature spores were pushed to the center while vegetative pathogens were in the surface layer of the xenoma. The V. necatrix meront usually contained two to three nuclei, and sporont contained two nuclei and was wrapped by a thick membrane with high electron density. The V. necatrix sporogony produces two types of spores, the ordinary dikaryotic spore and unicellular octospores, the latter of which were smaller in size and packed in a sporophorous vesicle. In summary, V. necatrix xenoma is a specialized cyst likely formed by fusion of multiple muscle cells and provides high concentration of energy and nutrients with increased number of mitochondria and endoplasmic reticulum for the massive proliferation of pathogens inside.

Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, we asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals.

Vitellogenin in the honey bee midgut

The alimentary canal carries out many functions critical to insect physiology, including digesting and absorbing nutrients and water, housing beneficial gut bacteria, and eliciting immunological responses. The midgut, in particular, is a compartment for digestion and absorption and serves as a first point of contact between ingested pathogens and the insect immune system. Recently, we found the protein vitellogenin (Vg) localized in midgut cells of some honey bee workers. Vg is an important egg-yolk precursor protein in nearly all oviparous animals, but it also has immunological functions shared across many taxa. Additional and unexpected Vg functions have been characterized in honey bees, but none of these functions involve the midgut directly. Therefore, we sought to map out how Vg is localized and expressed in this organ across the two most common worker bee behavioral groups, namely nurses and foragers. We used immunohistochemistry and quantitative reverse transcription PCR to show Vg has different localization patterns in nurses and foragers and limited gene expression. Our study provides a platform for building a more detailed understanding of the possible roles of Vg in insect midgut cells, and it adds to the current knowledge of this fascinating, multi-functional protein.

Characteristics of the Peritrophic Matrix of the Silkworm, Bombyx mori and Factors Influencing Its Formation.

Simple SummaryThe insect midgut is an important digestive organ with the peritrophic matrix (PM) being a semi-permeable membrane secreted by the midgut cells. The PM plays an important role in improving midgut digestion efficiency and protecting the midgut from food particles and exogenous pathogens. The silkworm, Bombyx mori, is an economically important insect. Understanding the structure of the PM is necessary for studying its function, but characteristics of PM in B. mori have been rarely reported. In this study, we conducted a comprehensive study on the PM structure of the PM in silkworms and found its thickness increased gradually during growth, but there was no difference in the thickness comparing the anterior, middle, and posterior regions. Permeability of the PM gradually decreased from the anterior to posterior regions. In addition, we found the formation of the PM was influenced by food ingestion and the gut microbiota.The peritrophic matrix (PM) secreted by the midgut cells of insects is formed by the binding of PM proteins to chitin fibrils. The PM envelops the food bolus, serving as a barrier between the content of the midgut lumen and its epithelium, and plays a protective role for epithelial cells against mechanical damage, pathogens, toxins, and other harmful substances. However, few studies have investigated the characteristics and synthesis factors of the PM in the silkworm, Bombyx mori. Here, we examined the characteristics of the PM in the silkworms. The PM thickness of the silkworms increased gradually during growth, while there was no significant difference in thickness along the entire PM region. Permeability of the PM decreased gradually from the anterior to posterior PM. We also found that PM synthesis was affected by food ingestion and the gut microbiota. Our results are beneficial for future studies regarding the function of the PM in silkworms.

Cr(VI)-induced DNA damage is lessened by the modulation of hsp70 via increased GSH de novo synthesis in Drosophila melanogaster.

Hexavalent chromium [Cr(VI)] is a genotoxic chemical, and in the chemical-exposed organism, oxidative stress is one of the leading causative mechanisms of genotoxicity. Heat shock protein-70 (Hsp70) is reported to be modulated in environmental chemical exposed organisms. Inadequate information on the protective role of Hsp70 in chemical-induced DNA lesions prompted us to investigate this possibility in a well-studied genetically tractable in vivo model Drosophila melanogaster. In the midgut cells of Cr(VI)-exposed hsp70-knockout (KO), -knockdown (KD), and -overexpression Drosophila strains, no significant change in double-strand breaks generation was observed in comparison to similarly exposed w 1118 and the respective genetic control strain after 48 h. Therefore, the role of hsp70 was investigated on oxidative DNA damage induction in the exposed organisms after 24 h. Oxidized DNA lesions (particularly oxidized purine-based lesions), 8-oxo-dG level, and oxidative stress endpoints were found to be significantly elevated in hsp70-KO and -KD strains in comparison to similarly exposed w 1118 and respective genetic control strain. On the contrary, in ubiquitous hsp70-overexpression strain exposed to Cr(VI), these endpoints were significantly lowered concurrently with increased GSH level through elevated gclc, and gclm expression, Gclc level, and GCL activity. The study suggests that as a consequence of hsp70 overexpression, the augmented GSH level in cells vis-a-vis GSH de novo synthesis can counteract Cr(VI)-induced oxidized DNA lesions.

Mesenchymal-to-epithelial transitions require tissue-specific interactions with distinct laminins.

Mesenchymal-to-epithelial transition (MET) converts cells from migratory mesenchymal to polarized epithelial states. Despite its importance for both normal and pathological processes, very little is known about the regulation of MET in vivo. Here we exploit midgut morphogenesis in Drosophila melanogaster to investigate the mechanisms underlying MET. We show that down-regulation of the EMT transcription factor Serpent is required for MET, but not sufficient, as interactions with the surrounding mesoderm are also essential. We find that midgut MET relies on the secretion of specific laminins via the CopII secretory pathway from both mesoderm and midgut cells. We show that secretion of the laminin trimer containing the Wingblister α-subunit from the mesoderm is an upstream cue for midgut MET, leading to basal polarization of αPS1 integrin in midgut cells. Polarized αPS1 is required for the formation of a monolayered columnar epithelium and for the apical polarization of αPS3, Baz, and E-Cad. Secretion of a distinct LamininA-containing trimer from midgut cells is required to reinforce the localization of αPS1 basally, and αPS3 apically, for robust repolarization. Our data suggest that targeting these MET pathways, in conjunction with therapies preventing EMT, may present a two-pronged strategy toward blocking metastasis in cancer.

Cp1/cathepsin L is required for autolysosomal clearance in Drosophila

ABSTRACT Macroautophagy/autophagy is a highly conserved lysosomal degradative pathway important for maintaining cellular homeostasis. Much of our current knowledge of autophagy is focused on the initiation steps in this process. Recently, an understanding of later steps, particularly lysosomal fusion leading to autolysosome formation and the subsequent role of lysosomal enzymes in degradation and recycling, is becoming evident. Autophagy can function in both cell survival and cell death, however, the mechanisms that distinguish adaptive/survival autophagy from autophagy-dependent cell death remain to be established. Here, using proteomic analysis of Drosophila larval midguts during degradation, we identify a group of proteins with peptidase activity, suggesting a role in autophagy-dependent cell death. We show that Cp1/cathepsin L-deficient larval midgut cells accumulate aberrant autophagic vesicles due to a block in autophagic flux, yet later stages of midgut degradation are not compromised. The accumulation of large aberrant autolysosomes in the absence of Cp1 appears to be the consequence of decreased degradative capacity as they contain undigested cytoplasmic material, rather than a defect in autophagosome-lysosome fusion. Finally, we find that other cathepsins may also contribute to proper autolysosomal degradation in Drosophila larval midgut cells. Our findings provide evidence that cathepsins play an essential role in the autolysosome to maintain basal autophagy flux by balancing autophagosome production and turnover. Abbreviations: 26-29-p: 26-29kD-proteinase; ADCD: autophagy-dependent cell death; Atg8a: Autophagy-related protein 8a; Cp1/cathepsin L: Cysteine proteinase-1; CtsB: Cathepsin B; cathD: cathepsin D; CtsF: Cathepsin F; GFP: green fluorescent protein; LAMP1: lysosomal-associated membrane protein 1; Mitf: microphthalmia associated transcription factor; PCA: principal component analysis; RNAi: RNA interference; RPF: relative to puparium formation

Characterizing the Honey Bee Interactome using Mass Spectrometry‐Based Proteomics

Western honey bees (Apis mellifera), as a superorganism, have an essential ecological and economic role as pollinators. However, various understudied pathogens infecting honey bees pose a significant threat to their health. One such pathogen is Nosema ceranae, a microsporidian parasite which rapidly proliferates in the honey bee ventriculus (midgut) cells, weakening their immune systems by competing for nutrients. Upon completing their lifecycle in the midgut cells, Nosema spores rupture host cells and spread into the rest of the gut and subsequently to other bees in the colony. The resulting symptoms of the diseased colony have been attributed to colony death. We hypothesize that host-pathogen interactions underpin Nosema infection in honey bees, and therefore understanding pathological protein interactions will suggest tools for combating Nosema. To this end, we used an optimized version of the mass spectrometry-based co-fractionation experiment described in Kristensen et al. (2012) This method uses size exclusion chromatography (SEC) to separate protein complexes into fractionated samples. After measuring protein amounts in each fraction through mass spectrometry, a machine learning pipeline detects proteins with similar separation profiles as protein interactions (Stacey et al., 2017). We can then assess the effects of Nosema infection. Using an in vivo approach, we first constructed a honey bee interactome from uninfected midgut cells. This is the first honey bee interactome to date. Validation experiments using co-immunoprecipitation coupled with mass spectrometry (IP-MS) demonstrate that this interactome is high quality and will be a valuable tool to future researchers. Using this interactome as a baseline, we can then compare interactomes derived from infected honey bee cells to identify the protein-based mechanisms of Nosema infection. Preliminary results from this highlight interactome changes in various processes such as peroxiredoxin activity and carbohydrate binding. This work presents the first honey bee interactome map. In addition, we are gaining novel insight into how protein interactions change upon infection.

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 microRNA (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 the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to those of mosquitoes infected with 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.

Accelerated delivery of dsRNA in lepidopteran midgut cells by a Galanthus nivalis lectin (GNA)-dsRNA-binding domain fusion protein

Lepidopteran insects are highly refractory to oral RNA interference (RNAi). Degradation, impaired cellular uptake and intracellular transport of double-stranded RNA (dsRNA) are considered the major factors responsible for the reduced RNAi efficiency in these insects. In this study, the potential of lectins to improve dsRNA delivery and RNAi efficacy was evaluated. First, a fusion protein consisting of the Galanthus nivalis agglutinin (GNA) and a dsRNA binding domain was developed, further referred to as GNA:dsRBD (GNAF). Then, its ability to increase dsRNA uptake and transfection efficiency in lepidopteran midgut cells was evaluated, as well as its ability to protect and promote the RNAi response in the beet armyworm Spodoptera exigua. Confocal microscopy analysis showed that GNAF-complexed dsRNA was internalized faster in Choristoneura fumiferana midgut CF1 cells (1 min) compared to naked dsRNA (>1 h). The faster uptake was also correlated with an increased RNAi efficiency in these CF1 cells. In vivo feeding bioassays with GNAF-complexed dsRNA led to an increased mortality in S. exigua compared to the controls. By targeting the essential gene V-ATPase A, we observed that the mortality increased to 48% in the GNAF-dsRNA treatment compared to only 8.3% and 6.6% in the control treatments with the naked dsRNA and the GNAF, respectively.

The Fate of Dietary Cholesterol in the Kissing Bug Rhodnius prolixus.

Insects are unable to synthesize cholesterol and depend on the presence of sterols in the diet for cell membrane composition and hormone production. Thus, cholesterol absorption, transport, and metabolism are potential targets for vector and pest control strategies. Here, we investigate the dietary cholesterol absorption and tissue distribution in the kissing bug Rhodnius prolixus using radiolabeled cholesterol. Both the anterior and posterior midguts absorbed cholesterol from the ingested blood, although the anterior midgut absorbed more. We also observed esterified cholesterol labeling in the epithelium, indicating that midgut cells can metabolize and store cholesterol. Only a small amount of labeled cholesterol was found in the hemolymph, where it was mainly in the free form and associated with lipophorin (Lp). The fat body transiently accumulated cholesterol, showing a labeled cholesterol peak on the fifth day after the blood meal. The ovaries also incorporated cholesterol, but cumulatively. The insects did not absorb almost half of the ingested labeled cholesterol, and radioactivity was present in the feces. After injection of 3H-cholesterol-labeled Lp into females, a half-life of 5.5 ± 0.7 h in the hemolymph was determined. Both the fat body and ovaries incorporated Lp-associated cholesterol, which was inhibited at low temperature, indicating the participation of active cholesterol transport. These results help describe an unexplored part of R. prolixus lipid metabolism.