Midgut Lumen Research Articles

Imidacloprid‐associated toxicity in the midgut of Sarcophaga ruficornis: Exploring histopathological, ultrastructural and biochemical alterations

AbstractThis study investigates the impact of imidacloprid insecticide, on the flesh fly Sarcophaga ruficornis, (Diptera: Sarcophagidae), a medically and veterinary significant species associated with myiasis. Lethal (0.02%) and sub‐lethal (0.004%) doses were administered over 24 and 48 h, focusing on midgut histopathological, ultrastructural and biochemical alterations. Imidacloprid‐induced disruptions in physiological and metabolic functions, leading to adult fly mortality. Histological and cytological analyses revealed significant cellular changes, including cell degeneration, cytoplasmic vacuolization, chromatin condensation, irregular epithelial borders, disrupted peritrophic membrane and the release of cell components into the midgut lumen. Biochemical analyses demonstrated a decrease in total protein, carbohydrate and lipid contents, correlating with insecticide concentration and duration. Antioxidant enzymes, particularly glutathione S‐transferase (GST) and superoxide dismutase (SOD), resulted in a significant increase compared to the control, proportional to time and concentration. This research provides a comprehensive understanding of imidacloprid‐induced toxicity in the midgut of Sarcophaga ruficornis, emphasizing its potential for effective pest population management.

Endochitinase from the Microsporidia Nosema ceranae facilitates infection in the honey bee Apis mellifera

Bees are crucial pollinators in terrestrial ecosystems, responsible for 80% of insect-driven pollination and playing a vital role in the pollination of 75% of crops. The honey bee, Apis mellifera, is not only used in honey production but also serves as a pollinator in agriculture. However, A. mellifera faces various challenges, including exposure to pathogens such as the Microsporidia Nosema ceranae, which has been linked to decreased crop yields and colony losses. Nosema ceranae spores infect adult honey bees by penetrating the midgut lumen and invading the cytoplasm of epithelial cells, completing their life cycle. However, the midgut possesses a protective mechanical barrier called the peritrophic matrix, composed of chitin and proteins, which prevents epithelial infection. Nevertheless, N. ceranae overcomes this primary defense mechanism, though the specific mechanisms it employs to cross the peritrophic matrix and reach the midgut epithelium are not yet well understood. This study aimed to investigate the potential role of the predicted endochitinase from N. ceranae to infect bees. We tested the hypothesis that inhibiting the expression of N. ceranae endochitinase through RNA interference would impact the pathogen infection of A. mellifera. Bees treated with dsRNA targeting endochitinase, administered 12 and 24 h after spore inoculation, exhibited suppressed endochitinase gene expression and a decrease in the number of total and viable N. ceranae spores in the midgut. These results indicate that inhibiting the expression of the target gene through RNA interference affects Microsporidia infection, underscoring the importance of this enzyme in the infection process.

CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm

Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.

Are bioplastics safe? Hazardous effects of polylactic acid (PLA) nanoplastics in Drosophila

The expanded uses of bioplastics require understanding the potential health risks associated with their exposure. To address this issue, Drosophila melanogaster as a versatile terrestrial in vivo model was employed, and polylactic acid nanoplastics (PLA-NPLs), as a proxy for bioplastics, were tested as a material model. Effects were determined in larvae exposed for 4 days to different concentrations (25, 100, and 400 μg/mL) of 463.9 ± 129.4 nm PLA-NPLs. Transmission electron microscopy (TEM) and scanning electron microscope (SEM) approaches permitted the detection of PLA-NPLs in the midgut lumen of Drosophila larvae, interacting with symbiotic bacteria. Enzymatic vacuoles were observed as carriers, collecting PLA-NPLs and enabling the crossing of the peritrophic membrane, finally internalizing into enterocytes. Although no toxic effects were observed in egg-to-adult survival, cell uptake of PLA-NPLs causes cytological disturbances and the formation of large vacuoles. The translocation across the intestinal barrier was demonstrated by their presence in the hemolymph. PLA-NPL exposure triggered intestinal damage, oxidative stress, DNA damage, and inflammation responses, as evaluated via a wide set of marker genes. Collectively, these structural and molecular interferences caused by PLA-NPLs generated high levels of oxidative stress and DNA damage in the hemocytes of Drosophila larvae. The observed effects point out the need for further studies aiming to deepen the health risks of bioplastics before adopting their uses as a safe plastic alternative.

Midgut health of Apis mellifera workers on oral administration of Lactobacillus rhamnosus and organic acids

A study about the provision of Lactobacillus rhamnosus NR_113332) and organic acids was carried out. 108 colony-forming units of probiotics were provided to the caged workers with and without the addition of 2.99% lactic acid, 2.91% acetic acid, and 1.96% acetic acid. After two weeks of oral administration, on termination, five bees were collected and dissected from each cage, and their midguts were cut into 8 μm thick sections by microtome. Subsequently, these histologically processed preparations were observed under camera fitted microscope. It was observed that the experimental workers were provided with diets: (pollen+ 50% w/v sucrose in 1.96% acetic acid, pollen+ L. rhamnosus in 50% w/v sucrose in distilled water, pollen+ L. rhamnosus in 50% w/v sucrose in 2.99% lactic acid, pollen+ L. rhamnosus in 50% w/v sucrose in 2.91% acetic acid, pollen+ L. rhamnosus in 50% w/v sucrose in 1.96% acetic acid) had a considerable quantity of peritrophic membranes. The mean midgut lumen diameters (µm2) were 373.33 ± 98.38, 296.67 ± 23.33, 243.33 ± 71.72, 426.67 ± 40.55, 576.67 ± 93.87 as compared to control’s 240.00 ± 30.55. The provision of probiotic and organic acids resulted in a slightly disturbed midgut epithelial wall, the presence of more quantity of peritrophic membranes, saved regeneration crypts, more gastric cells discharging, and some cytoplasmic vacuolization of caged worker bees. Our study suggests that probiotics and organic acids can be used as an environmentally friendly apipromotor, but verification by field studies is needed.

Post-feeding transcriptomics reveals essential genes expressed in the midgut of the desert locust.

The digestive tract constitutes an important interface between an animal's internal and external environment. In insects, available gut transcriptome studies are mostly exploratory or look at changes upon infection or upon exposure to xenobiotics, mainly performed in species belonging to holometabolan orders, such as Diptera, Lepidoptera or Coleoptera. By contrast, studies focusing on gene expression changes after food uptake and during digestion are underrepresented. We have therefore compared the gene expression profiles in the midgut of the desert locust, Schistocerca gregaria, between three different time points after feeding, i.e., 24h (no active digestion), 10min (the initial stage of feeding), and 2h (active food digestion). The observed gene expression profiles were consistent with the polyphagous herbivorous lifestyle of this hemimetabolan (orthopteran) species. Our study reveals the upregulation of 576 genes 2h post-feeding. These are mostly predicted to be associated with digestive physiology, such as genes encoding putative digestive enzymes or nutrient transporters, as well as genes putatively involved in immunity or in xenobiotic metabolism. The 10min time point represented an intermediate condition, suggesting that the S. gregaria midgut can react rapidly at the transcriptional level to the presence of food. Additionally, our study demonstrated the critical importance of two transcripts that exhibited a significant upregulation 2h post-feeding: the vacuolar-type H(+)-ATPase and the sterol transporter Niemann-Pick 1b protein, which upon RNAi-induced knockdown resulted in a marked increase in mortality. Their vital role and accessibility via the midgut lumen may make the encoded proteins promising insecticidal target candidates, considering that the desert locust is infamous for its huge migrating swarms that can devastate the agricultural production in large areas of Northern Africa, the Middle East, and South Asia. In conclusion, the transcriptome datasets presented here will provide a useful and promising resource for studying the midgut physiology of S. gregaria, a socio-economically important pest species.

Requirement of Snakeskin for normal functions of midgut and Malpighian tubules in Henosepilachna vigintioctopunctata.

Septate junctions (SJs) are located between epithelial cells and play crucial roles in epithelial barrier formation and epithelia cell homeostasis. Nevertheless, the molecular constituents, especially those related to smooth SJs (sSJs), have not been well explored in non-Drosophilid insects. A putative integral membrane protein Snakeskin (Ssk) was identified in a Coleoptera foliar pest Henosepilachna vigintioctopunctata. RNA interference-aided knockdown of Hvssk at the third-instar larval stage arrested larval development. Most resultant larvae failed to shed larval exuviae until their death. Silence of Hvssk at the fourth-instar larvae inhibited the growth and reduced foliage consumption. Dissection and microscopic observation revealed that compromised expression of Hvssk caused obvious phenotypic defects in the midgut. A great number of morphologically abnormal columnar epithelial cells accumulated throughout the midgut lumen. Moreover, numerous vesicles were observed in the malformed cells of the Malpighian tubules (Mt). All the Hvssk depleted larvae remained as prepupae; they gradually darkened and eventually died. Furthermore, depletion of Hvssk at the pupal stage suppressed adult feeding and shortened adult lifespan. These findings demonstrated that Ssk plays a vital role in the integrity and function of both midguts and Mt, and established the conservative roles of Ssk in the formation of epithelial barrier and the homeostasis of epithelial cells in H. vigintioctopunctata.

Optimized high-yield preparation of alkaline-solubilizable crystalline inclusion of the Bacillus thuringiensis Cry4Aa δ-endotoxin expressed in Escherichia coli

The native Cry4Aa δ-endotoxin produced exclusively in Bacillus thuringiensis during sporulation as a ∼130-kDa inactive protoxin is confined within the parasporal crystalline inclusion that dissolves at alkaline pH in the midgut lumen of mosquito larvae. Here, the recombinant Cry4Aa toxin over-expressed in Escherichia coli at 30 °C as an alkaline-solubilizable inclusion was found inevitably lost during isolation from the cell lysate (pH ∼6.5) of which host cells were pre-suspended in distilled water (pH ∼5.5). When 100 mM KH2PO4 (pH 5.0) was used as host cell-suspending buffer, the cell lysate's pH became more acidic (pH 5.5), allowing the expressed protoxin to be entirely retained in the form of crystalline inclusion rather than a soluble form, and thus high-yield recovery of the partially purified inclusion was obtained. Upon dialysis of the alkaline-solubilized protoxin against the KH2PO4 buffer, the protoxin precipitate was efficiently recovered and still exhibited high toxicity to Aedes aegypti mosquito larvae. Additionally, the precipitated protoxin was completely resolubilized in 50 mM Na2CO3 buffer (pH 9.0) and proteolytically processed by trypsin to produce the 65-kDa activated toxin comprising ∼47- and ∼20-kDa fragments. In silico structural analysis suggested that His154, His388, His536 and His572 were involved in a dissolution of the Cry4Aa inclusion at pH 6.5, conceivably through interchain salt bridge breakage. Altogether, such an optimized protocol described herein was effective for the preparation of alkaline-solubilizable inclusions of the recombinant Cry4Aa toxin in large amounts (>25 mg per liter culture) that would pave the way for further structure-function relationship studies of different Cry toxins.

Steroid hormone-dependent changes in trehalose physiology in the silkworm, Bombyx mori.

Holometabolous insects undergo metamorphosis to reconstruct their body to the adult form during pupal period. Since pupae cannot take any diets from the outside because of a hard pupal cuticle, those insects stock up on nutrients sufficient for successful metamorphosis during larval feeding period. Among those nutrients, carbohydrates are stored as glycogen or trehalose, which is the major blood sugar in insects. The hemolymph trehalose is constantly high during the feeding period but suddenly decreases at the beginning of the prepupal period. It is believed that trehalase, which is a trehalose-hydrolyzing enzyme, becomes highly active to reduce hemolymph trehalose level during prepupal period. This change in the hemolymph trehalose level has been interpreted as the physiological shift from storage to utilization of trehalose at that stage. Although this shift in trehalose physiology is indispensable for energy production required for successful metamorphosis, little is known on the regulatory mechanisms of trehalose metabolism in accordance with developmental progress. Here, we show that ecdysone, an insect steroid hormone, plays essential roles in the regulation of soluble trehalase activity and its distribution in the midgut of silkworm, Bombyx mori. In the end of larval period, soluble trehalase was highly activated in the midgut lumen. This activation was disappeared in the absence of ecdysone and also restored by ecdysone administration. Our present results suggest that ecdysone is essentially required for the changes in the function of the midgut on trehalose physiology as development progresses.

Dietary compounds activate an insect gustatory receptor on enteroendocrine cells to elicit myosuppressin secretion.

Sensing of midgut internal contents is important for ensuring appropriate hormonal response and digestion following the ingestion of dietary components. Studies in mammals have demonstrated that taste receptors (TRs), a subgroup of G protein-coupled receptors (GPCRs), are expressed in gut enteroendocrine cells (EECs) to sense dietary compounds and regulate the production and/or secretion of peptide hormones. Although progress has been made in identifying expression patterns of gustatory receptors (GRs) in gut EECs, it is currently unknown whether these receptors, which act as ligand-gated ion channels, serve similar functions as mammalian GPCR TRs to elicit hormone production and/or secretion. A Bombyx mori Gr, BmGr6, has been demonstrated to express in cells by oral sensory organs, midgut and nervous system; and to sense isoquercitrin and chlorogenic acid, which are non-nutritional secondary metabolites of host mulberry. Here, we show that BmGr6 co-expresses with Bommo-myosuppressin (BMS) in midgut EECs, responds to dietary compounds and is involved in regulation of BMS secretion. The presence of dietary compounds in midgut lumen after food intake resulted in an increase of BMS secretions in hemolymph of both wild-type and BmGr9 knockout larvae, but BMS secretions in BmGr6 knockout larvae decreased relative to wild-type. In addition, loss of BmGr6 led to a significant decrease in weight gain, excrement, hemolymph carbohydrates levels and hemolymph lipid levels. Interestingly, although BMS is produced in both midgut EECs and brain neurosecretory cells (NSCs), BMS levels in tissue extracts suggested that the increase in hemolymph BMS during feeding conditions is primarily due to secretion from midgut EECs. Our studies indicate that BmGr6 expressed in midgut EECs responds to the presence of dietary compounds in the lumen by eliciting BMS secretion in B. mori larvae.

Silencing of the immune gene BmPGRP-L4 in the midgut affects the growth of silkworm (Bombyx mori) larvae.

Peptidoglycan recognition proteins (PGRPs) are one of the receptors in insects' immune pathways, essential for insects to recognize the exogenous pathogens in order to activate the Toll and immune deficiency (IMD) pathway. In the silkworm Bombyx mori, previous studies focused on the short PGRPs and less is known about the long PGRPs. In this study, a long PGRP in silkworm BmPGRP-L4 was cloned and its expression and function were analysed. The results showed that BmPGRP-L4 contains a transmembrane region, a conserved PGRP domain, and an amidase-2 domain. The expression profile demonstrated that BmPGRP-L4 existed in diverse tissues including epidermis, fat body, midgut, and silk glands, with remarkably high expression in the midgut in the 5th instar. Oral infection with Escherichia coli and Staphylococcus aureus significantly induced BmPGRP-L4 in the midgut and epidermis, as well as in the fat body and silk glands. Peptidoglycan also induced the expression of BmPGRP-L4 in midgut tissue ex vivo and BmN4 cells in vitro. RNAi of BmPGRP-L4 was effective in the midgut and epidermis, while the efficiency in the fat body was transient. RNAi-mediated knock-down of BmPGRP-L4 reduced the weight and growth of the silkworm, possibly due to its participation in the immune response and the regulation of the microbiota in the midgut lumen of the silkworm larvae.

Trypsin, the Major Proteolytic Enzyme for Blood Digestion in the Mosquito Midgut.

When a female mosquito takes a blood meal, proteolytic activity surges in the midgut. Trypsin-like serine proteases are the major endoproteolytic enzyme induced by feeding in mosquitoes. The mosquito midgut lacks trypsin activity before the blood meal, but in most anautogenous mosquitoes, trypsin activity increases continuously up to 30 h after feeding and subsequently returns to baseline levels by 60 h. Trypsin activity in mosquitoes is restricted entirely to the posterior midgut lumen, where blood is stored and digested. Trypsin enzyme activity can be quantitatively measured using the artificial Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride substrate, a method described in our associated protocol.

The peritrophic matrix delays Nosema ceranae infection in the honey bee Apis mellifera midgut

AbstractThe midgut of bees plays a significant role in digestion and absorption and is a gateway for pathogens. As a protection for the organism, the midgut lumen is lined by a peritrophic matrix rich in chitin fibrils, associated with proteins that form multiple resistant layers with gelatinous consistency, with several functions, including the mechanical barrier against infection by pathogens. The obligate intracellular microsporid Nosema ceranae is an important pathogen that decreases honey production, increases pesticide susceptibility, and may cause mortality in the honey bee Apis mellifera. This study evaluated whether the peritrophic matrix has a protective function against infection by N. ceranae in A. mellifera adult workers. The peritrophic matrix integrity was changed by feeding the bees with calcofluor white (CFW), followed by an artificial infection with N. ceranae spores. Results shown that 0.5% CFW affect the integrity of the peritrophic matrix with areas of interruption of this envelope. This disorganization of the peritrophic matrix reduces the life cycle period of N. ceranae from eight to four days, after inoculation, and increases the concentration of new spores released in the midgut lumen. This is the first report on the importance of the peritrophic matrix as a protective barrier against N. ceranae infection in A. mellifera.

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.

Development, gut health, and longevity of European bee on the provision of biosugar syrup

Syrup containing monomeric sugars was prepared from banana-peel waste by hydrolyzing it with cellulose as well as pectinase to feed honeybees instead of using sucrose syrup in the period when there is no nectar in flowers. This syrup was provided to the caged workers with and without the addition of monomeric sugars (glucose, fructose) for a period of two weeks. It was observed that worker bees belonging to experiment No. 2 (pollens + 25% w/v glucose in BPW sugar syrup) had a significant increase in longevity. In addition to this, morphometric and histological analyses were also done. Morphometric studies confirmed reasonable increases in different body parameters of worker bees. Their hypopharyngeal gland became developed with a mean acinal surface value of 0.019 ± 0.0 mm2 as compared to the control's 0.0124 ± 0.0 mm2. Sections of the midgut (stomach) and small intestine up to 8 μm were prepared and observed under camera fitted microscope. Bees of experiment No. 2 had peritrophic membranes in close contact with rabdorium-like control bee's midgut, nuclei stained hyper chromatic, considerable increase in gastric cells discharging, and compact and homogeneous mass of semi-digested pollens in the central part of the intestinal lumen which indicates the improved digestion. The mean midgut lumen diameter was 450 ± 34.6 μm2 as compared to the control's 240.0 ± 30.5 μm2 and the mean intestinal lumen diameter was 223.3 ± 33.3 μm2 as compared to the control's 113.3 ± 38.4 μm2 which is an indication of tissue development. In the end, it is concluded that the use of biosugar syrup is good alternative to sucrose sugar syrup for honeybees.

Tick transmission of Borrelia burgdorferi to the murine host is not influenced by environmentally acquired midgut microbiota

BackgroundIxodes scapularis is the predominant tick vector of Borrelia burgdorferi, the agent of Lyme disease, in the USA. Molecular interactions between the tick and B. burgdorferi orchestrate the migration of spirochetes from the midgut to the salivary glands—critical steps that precede transmission to the vertebrate host. Over the last decade, research efforts have invoked a potential role for the tick microbiome in modulating tick-pathogen interactions.ResultsUsing multiple strategies to perturb the microbiome composition of B. burgdorferi-infected nymphal ticks, we observe that changes in the microbiome composition do not significantly influence B. burgdorferi migration from the midgut, invasion of salivary glands, or transmission to the murine host. We also show that within 24 and 48 h of the onset of tick feeding, B. burgdorferi spirochetes are within the peritrophic matrix and epithelial cells of the midgut in preparation for exit from the midgut.ConclusionsThis study highlights two aspects of tick-spirochete interactions: (1) environmental bacteria associated with the tick do not influence spirochete transmission to the mammalian host and (2) the spirochete may utilize an intracellular exit route during migration from the midgut to the salivary glands, a strategy that may allow the spirochete to distance itself from microbiota in the midgut lumen effectively. This may explain in part, the inability of environment-acquired midgut microbiota to significantly influence spirochete transmission. Unraveling a molecular understanding of this exit strategy will be critical to gain new insights into the biology of the spirochete and the tick.7AdntFwsX5JjuXz-jKBq7oVideo

Larval and adult digestive tract of the carrion beetle Oxelytrum discicolle (Brullé, 1840) (Coleoptera: Silphidae)

Oxelytrum discicolle is a necrophagous beetle occurring in Central and South America, with potential use in forensic entomology for investigations in the context of legal medicine. The present work aimed to describe the morphology of the digestive tract of larvae and adults and contributes to the knowledge about the digestion associated with necrophagy. In the three larval instars, the foregut is short and narrow, the midgut is a dilated tube, elongated with a smooth surface, and the hindgut is narrow and long, with small lateral projections and a dilated terminal region. The gut epithelium in the second and third instar larvae is vacuolated in the mid- and hindgut, with high production of apocrine secretions in the midgut lumen. In adults, the foregut is short, with small spines in the cuticular intima that covers the flattened epithelium. The midgut is dilated, with many short gastric caeca with regenerative cells in the blind portion, which differentiate in digestive columnar cells towards the midgut lumen. The hindgut is long and narrow, with an enlarged distal portion with folded epithelium lined by a thin cuticle. The histochemical tests reveal the absence of protein storage granules in the gut epithelium of larvae and adults. The gut characteristics of the carrion beetle O. discicolle are similar to those of other predatory and phytophagous Coleoptera, which may indicate that ancestry may influence the alimentary canal morphology more than the feeding habits.

Azole selenourea disrupted the midgut and caused malformed development of Plutella xylostella

Chemical insecticides targeting the digestive system of diamondback moth (DBM), Plutella xylostella, have not been developed. The discovery of an insecticide with novel mode of action is a challenge for the control of DBM. In this study, a class of selenium- and difluoromethyl-modified azoles (fluoroazole selenoureas, FASU) were designed and synthesized for the control of DBM. Of these azoles, compound B4 showed the highest insecticidal activity against DBM. The LC50 of third- and second-instar larvae reached 32.3 and 4.6 μg mL–1, respectively. The midgut tissue of larvae was severely disrupted, and the larval intestinal tissue was dotted with unique red spots after treatment with compound B4. Compound B4 led to disintegration of the peritrophic matrix, swelling of the midgut epithelium, fracture of the microvilli, and extensive leakage of cellular debris in the midgut lumen. Surviving larvae grew very slowly, and the larval duration was significantly prolonged after exposure to compound B4 at sublethal doses (LC10, LC25 and LC50). Furthermore, the pupation rate, emergence rate and pupae weight were significantly decreased. Compound B4 also induced abnormal pupae, causing adults to be trapped in the cocoon or failure to fly due to twisted wings. These results demonstrated that FASU could reduce the population of DBM in sublethal doses. FASU is the first synthetic insecticidal lead compound that has been shown to disrupt the midgut tissue of the larvae of DBM, and its mode of action totally differs from that of commercial chemical insecticides.

Impact of weathered multi-walled carbon nanotubes on the epithelial cells of the intestinal tract in the freshwater grazers Lymnaea stagnalis and Rhithrogena semicolorata

Freshwater grazers are suitable organisms to investigate the fate of environmental pollutants, such as weathered multi-walled carbon nanotubes (wMWCNTs). One key process is the uptake of ingested materials into digestive or absorptive cells. To address this, we investigated the localization of wMWCNTs in the intestinal tracts of the mud snail Lymnaea stagnalis (L. stagnalis) and the mayfly Rhithrogena semicolorata (R. semicolorata). In L. stagnalis, bundles of wMWCNTs could be detected in the midgut lumen, whereas only single wMWCNTs could be detected in the lumina of the digestive gland. Intracellular uptake of wMWCNTs was detected by transmission electron microscopy (TEM) but was restricted to the cells of the digestive gland. In larvae of R. semicolorata, irritations of the microvilli and damages in the apical parts of the epithelial gut cells were detected after feeding with 1 to 10 mg/L wMWCNTs. In both models, we detected fibrillar structures in close association with the epithelial cells that formed peritrophic membranes (PMs). The PM may cause a reduced transmission of wMWCNT bundles into the epithelium by forming a filter barrier and potentially protecting the cells from the wMWCNTs. As a result, the uptake of wMWCNTs into cells is rare in mud snails and may not occur at all in mayfly larvae. In addition, we monitor physiological markers such as levels of glycogen or triglycerides and the RNA/DNA ratio. This ratio was significantly affected in L. stagnalis after 24 days with 10 mg/L wMWCNTs, but not in R. semicolorata after 28 days and 10 mg/L wMWCNTs. However, significant effects on the energy status of R. semicolorata were analysed after 28 days of exposure to 1 mg/L wMWCNTs. Furthermore, we observed a significant reduction of phagosomes per enterocyte cell in mayfly larvae at a concentration of 10 mg/L wMWCNTs (p < 0.01).

Transgenic Anopheles mosquitoes expressing human PAI-1 impair malaria transmission

In mammals, the serine protease plasmin degrades extracellular proteins during blood clot removal, tissue remodeling, and cell migration. The zymogen plasminogen is activated into plasmin by two serine proteases: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), a process regulated by plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor that specifically inhibits tPA and uPA. Plasmodium gametes and sporozoites use tPA and uPA to activate plasminogen and parasite-bound plasmin degrades extracellular matrices, facilitating parasite motility in the mosquito and the mammalian host. Furthermore, inhibition of plasminogen activation by PAI-1 strongly blocks infection in both hosts. To block parasite utilization of plasmin, we engineered Anopheles stephensi transgenic mosquitoes constitutively secreting human PAI-1 (huPAI-1) in the midgut lumen, in the saliva, or both. Mosquitoes expressing huPAI-1 strongly reduced rodent and human Plasmodium parasite transmission to mosquitoes, showing that co-opting plasmin for mosquito infection is a conserved mechanism among Plasmodium species. huPAI-1 expression in saliva induced salivary gland deformation which affects sporozoite invasion and P. berghei transmission to mice, resulting in significant levels of protection from malaria. Targeting the interaction of malaria parasites with the fibrinolytic system using genetically engineered mosquitoes could be developed as an intervention to control malaria transmission.