Exposure to environmental pharmaceuticals affects the macromolecular composition of mussels digestive glands.

Human pharmaceuticals represent a major challenge in natural environment. A better knowledge on their mechanisms of action and adverse effects on cellular pathways is fundamental to predict long-term consequences for marine wildlife. The FTIRI Imaging (FTIRI) spectroscopy represents a vibrational technique allowing to map specific areas of non-homogeneous biological samples, providing a unique biochemical and ultrastructural fingerprint of the tissue. In this study, FTIRI technique has been applied, for the first time, to characterize (i) the chemical building blocks of digestive glands of Mytilus galloprovincialis, (ii) alterations and (iii) resilience of macromolecular composition, after a 14-days exposure to 0.5 µg/L of carbamazepine (CBZ), valsartan (VAL) and their mixture, followed by a 14-days recovery period. Spectral features of mussels digestive glands provided insights on composition and topographical distribution of main groups of biological macromolecules, such as proteins, lipids, and glycosylated compounds. Pharmaceuticals caused an increase in the total amount of protein and a significant decrease of lipids levels. Changes in macromolecular features reflected the modulation of specific molecular and biochemical pathways thus supporting our knowledge on mechanisms of action of such emerging pollutants. Overall, the applied approach could represent an added value within integrated strategies for the effects-based evaluation of environmental contaminants.

Proteotranscriptomic analyses of the midgut and Malpighian tubules after a sublethal concentration of Cry1Ab exposure on Spodoptera litura.

BACKGROUND: Cry1Ab has emerged as a bio-insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets. RESULTS: After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag-based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up-regulated and significantly enriched in the serine protease activity pathway, and up-regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down-regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein-protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt-resistance of S. litura larvae under sublethal Cry1Ab exposure. CONCLUSION: Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry.

A longitudinal transcriptomic analysis of Rhipicephalus microplus midgut upon feeding.

Rhipicephalus microplus, a highly host-specific tick that primarily feeds on cattle, posing a significant threat to livestock production. The investigation of tick physiology is crucial for identifying potential targets in tick control. Of particular interest adult female ticks undergo a significant expansion of the midgut during feeding, leading to an over 100-fold increase in body weight. Beyond the functions of storing and digesting blood meals, the tick midgut plays a crucial role in acquiring and transmitting pathogens. However, our understanding of tick midgut physiology remains limited. In this study we conducted a comprehensive longitudinal transcriptome analysis of the midgut from adult female R. microplus ticks collected at various feeding stages, providing an overview of the transcriptional modulation in this organ as feeding progress. By employing a de novo assembly approach followed by coding-sequences (CDS) extraction, 60,599 potential CDS were identified. In preparation for functional annotation and differential expression analysis, transcripts that showed an average transcript per million (TPM) ≥ 3 in at least one of the biological conditions were extracted. This selection process resulted in a total of 10,994 CDS, which were categorized into 24 functional classes. Notably, our differential expression analysis revealed three main transcriptional profiles. In the first one, representing the slow-feeding stage, the most abundant functional classes were the "protein synthesis" and "secreted" groups, reflecting the highly active state of the tick midgut. The second profile partially accounts for the rapid-feeding stage, in which a high number of differentially expressed transcripts was observed. Lastly, the third transcriptional profile represents post-detached ticks. Notably the highest number of modulated transcripts was observed up to 48 h post-detachment (hpd), however no major differences was observed up to 168 hpd. Overall, the data presented here offers a temporal insight into tick midgut physiology, contributing to the identification of potential targets for the development of anti-tick control strategies.

The digestive system of a cricket pulverizes polyethylene microplastics down to the nanoplastic scale.

Microplastics (MPs; <5 mm) are a growing concern and a poorly understood threat to biota. We used a generalist insect (a cricket; Gryllodes sigillatus) to examine whether individuals would ingest and physically degrade MPs in their food. We fed crickets a range of concentrations (0, 2.5, 5, and 10% w/w) of fluorescent polyethylene MPs mixed into a standard diet and dissected the gut regions to isolate the MPs within. Comparing plastic content and fragment size within gut regions, we sought to identify whether and where crickets can fragment ingested MP particles. Given the digestive tract morphology of this species, we expected that the crickets would both ingest and egest the MPs. We also predicted that the MPs would be fragmented into smaller pieces during this digestive process. We found that G. sigillatus egested much smaller pieces than they ingested, and this fragmentation occurs early in the digestive process of this insect. We found this for both sexes as well as across the range of concentrations of MPs. The degree of plastic breakdown relative to plastic feeding time suggests that the ability to fragment MPs is intrinsic and not altered by how much time crickets have spent eating the plastics. The amount of plastics found in each region of the gut in relation to feeding time also suggests that this size and shape of PE microplastic does not cause any physical blockage in the gut. This lack of evidence for blockage is likely due to plastic breakdown. We found a ∼1000-fold reduction in plastic size occurs during passage through the digestive system, yielding particles very near nanoplastics (NPs; <1 µm), and likely smaller, that are then excreted back into the environment. These findings suggest that generalist insects can act as agents of plastic transformation in their environment if/when encountering MPs.

A chemo-mechanical model of endoderm movements driving elongation of the amniote hindgut.

Although mechanical and biochemical descriptions of development are each essential, integration of upstream morphogenic cues with downstream tissue mechanics remains understudied during vertebrate morphogenesis. Here, we developed a two-dimensional chemo-mechanical model to investigate how mechanical properties of the endoderm and transport properties of fibroblast growth factor (FGF) regulate avian hindgut morphogenesis in a coordinated manner. Posterior endoderm cells convert a gradient of FGF ligands into a contractile force gradient, leading to a force imbalance that drives collective cell movements that elongate the forming hindgut tube. We formulated a 2D reaction-diffusion-advection model describing the formation of an FGF protein gradient as a result of posterior displacement of cells transcribing unstable Fgf8 mRNA during axis elongation, coupled with translation, diffusion and degradation of FGF protein. The endoderm was modeled as an active viscous fluid that generates contractile stresses in proportion to FGF concentration. With parameter values constrained by experimental data, the model replicates key aspects of hindgut morphogenesis, suggests that graded isotropic contraction is sufficient to generate large anisotropic cell movements, and provides new insight into how chemo-mechanical coupling across the mesoderm and endoderm coordinates hindgut elongation with axis elongation.

Biological function and clinical application prospect of tsRNAs in digestive system biology and pathology.

tsRNAs are small non-coding RNAs originating from tRNA that play important roles in a variety of physiological activities such as RNA silencing, ribosome biogenesis, retrotransposition, and epigenetic inheritance, as well as involvement in cellular differentiation, proliferation, and apoptosis. tsRNA-related abnormalities have a significant influence on the onset, development, and progression of numerous human diseases, including malignant tumors through affecting the cell cycle and specific signaling molecules. This review introduced origins together with tsRNAs classification, providing a summary for regulatory mechanism and physiological function while dysfunctional effect of tsRNAs in digestive system diseases, focusing on the clinical prospects of tsRNAs for diagnostic and prognostic biomarkers. Video Abstract.

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

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

Piezo1 in Digestive System Function and Dysfunction.

Piezo1, a non-selective cation channel directly activated by mechanical forces, is widely expressed in the digestive system and participates in biological functions physiologically and pathologically. In this review, we summarized the latest insights on Piezo1's cellular effect across the entire digestive system, and discussed the role of Piezo1 in various aspects including ingestion and digestion, material metabolism, enteric nervous system, intestinal barrier, and inflammatory response within digestive system. The goal of this comprehensive review is to provide a solid foundation for future research about Piezo1 in digestive system physiologically and pathologically.

The Haematopoietically-expressed homeobox transcription factor: roles in development, physiology and disease.

The Haematopoietically expressed homeobox transcription factor (Hhex) is a transcriptional repressor that is of fundamental importance across species, as evident by its evolutionary conservation spanning fish, amphibians, birds, mice and humans. Indeed, Hhex maintains its vital functions throughout the lifespan of the organism, beginning in the oocyte, through fundamental stages of embryogenesis in the foregut endoderm. The endodermal development driven by Hhex gives rise to endocrine organs such as the pancreas in a process which is likely linked to its role as a risk factor in diabetes and pancreatic disorders. Hhex is also required for the normal development of the bile duct and liver, the latter also importantly being the initial site of haematopoiesis. These haematopoietic origins are governed by Hhex, leading to its crucial later roles in definitive haematopoietic stem cell (HSC) self-renewal, lymphopoiesis and haematological malignancy. Hhex is also necessary for the developing forebrain and thyroid gland, with this reliance on Hhex evident in its role in endocrine disorders later in life including a potential role in Alzheimer's disease. Thus, the roles of Hhex in embryological development throughout evolution appear to be linked to its later roles in a variety of disease processes.

The roles of enteroendocrine cell distribution and gustatory receptor expression in regulating peptide hormone secretion in the midgut of Bombyx mori larvae.

To regulate physiological homeostasis and behavior in Bombyx mori, more than 20 peptide hormones in the midgut of larvae are secreted upon detection of food substances at the lumen. Although it is logical to assume that the timings of peptide hormone secretions are regulated, little is known about the mechanisms. In this study, the distributions of enteroendocrine cells (EECs) producing five peptide hormones and EECs expressing gustatory receptors (Grs), as candidate receptors for luminal food substances and nutrients, were examined via immunostaining in B. mori larvae. Three patterns of peptide hormone distribution were observed. Tachykinin (Tk)- and K5-producing EECs were located throughout the midgut; myosuppressin-producing EECs were located in the middle-to-posterior midgut; and allatostatin C- and CCHamide-2-producing EECs were located in the anterior-to-middle midgut. BmGr4 was expressed in some Tk-producing EECs in the anterior midgut, where food and its digestive products arrived 5 min after feeding began. Enzyme-linked immunosorbent assay (ELISA) revealed secretion of Tk starting approximately 5 min after feeding began, suggesting that food sensing by BmGr4 may regulate Tk secretion. BmGr6 was expressed in a few Tk-producing EECs in the middle-to-posterior midgut, although its significance was unclear. BmGr6 was also expressed in many myosuppressin-producing EECs in the middle midgut, where food and its digestive products arrived 60 min after feeding began. ELISA revealed secretion of myosuppressin starting approximately 60 min after feeding began, suggesting that food sensing by BmGr6 may regulate myosuppressin secretion. Finally, BmGr9 was expressed in many BmK5-producing EECs throughout the midgut, suggesting that BmGr9 may function as a sensor for the secretion of BmK5.

The Study of Alanine Transaminase Activity in Tissues of Silkworm (Bombyx mori) via Direct Analysis in Real-Time (DART) Mass Spectrometry.

Alanine transaminase (ALT) is an important amino acid-metabolizing enzyme in silkworm Bombyx mori L., and is mainly involved in transferring glutamate to alanine (serving as an essential precursor in silk protein synthesis) through transamination. Therefore, it is generally believed that silk protein synthesis in the silk gland and the cocoon quantity increase with the increase in ALT activity to a certain extent. Here, a novel analytical method was developed to determine the ALT activity in several key tissues of Bombyx mori L. including the posterior silk gland, midgut, fat body, middle silk gland, trachea and hemolymph, by combining the direct-analysis-in-real-time (DART) ion source with a triple-quadrupole mass spectrometer. In addition, a traditional ALT activity assay, the Reitman-Frankel method, was also used to measure ALT activity for comparison. The ALT activity results obtained via the DART-MS method are in good agreement with those obtained via the Reitman-Frankel method. However, the present DART-MS method provides a more convenient, rapid and environmentally friendly quantitative method for ALT measurement. Especially, this method can also monitor ALT activity in different tissues of Bombyx mori L. in real time.

The Role of Protein Arginine Methyltransferases in Pathogenesis and Treatment of Digestive System Carcinoma.

Posttranslational modification of proteins increases their diversity and maintains the stability of the intracellular environment. Protein arginine methyltransferases (PRMT) are an important family of epigenetic modification enzymes, which play a critical role in posttranslational modification. In recent years, with the in-depth study of the role of epigenetics, the structure and function of PRMTs have been gradually understood. PRMT enzymatic activity is related to a variety of cellular processes in digestive system malignancies, such as inflammation and immune response, activation of cell cycle and proliferation, inhibition of apoptosis, DNA damage repair, and epithelial-mesenchymal transition. A variety of chemical tools are developed to inhibit PRMT activity, which have been verified by tumor models and clinical trials. This review summarizes the structure and functions of PRMTs as a prelude to our further studies on their role in tumors. The involvement of different PRMTs in the pathogenesis of gastrointestinal tumors is then reviewed. In addition, the application of PRMT inhibitors as therapeutic agents for digestive system cancers is highlighted. In conclusion, PRMTs play an important role in the pathogenesis of gastrointestinal tumors, and their prognostic and therapeutic potential warrants further investigation.

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.

LINC00511, a future star for the diagnosis and therapy of digestive system malignant tumors.

The digestive system malignant tumors (DSMTs), mainly consist of digestive tract and digestive gland tumors, become an inescapable culprit to hazard human health worldwide. Due to the huge hysteresis in the cognitive theories of DSMTs occurrence and progression, advances in medical technology have not improved the prognosis. Therefore, more studies on a variety of tumor-associated molecular biomarkers and more detailed disclosure on potential regulatory networks are urgently needed to facilitate the diagnostic and therapeutic strategies of DSMTs. With the development of cancer bioinformatics, a special type of endogenous RNA involved in multi-level cellular function regulation rather than encoding protein, is categorized as non-coding RNAs (ncRNAs) and becomes a hotspot issue in oncology. Among them, long non-coding RNAs (lncRNAs), transcription length > 200 nt, show obvious superiority in both research quantity and dimension compared to microRNAs (miRNAs) and circular RNAs (circRNAs). As a recently discovered lncRNA, LINC00511 has been confirmed to be closely associated with DSMTs and might be exploited as a novel biomarker. In the present review, the comprehensive studies of LINC00511 in DSMTs are summarized, as well as the underlying molecular regulatory networks. In addition, deficiencies in researches are point out and discussed. The Cumulative oncology studies provide a fully credible theoretical basis for identifying the regulatory role of LINC00511 in human DSMTs. LINC00511, proved to be an oncogene in DSMTs, might be defined as a potential biomarker for diagnosis and prognosis evaluation, as well as a rare therapeutic target.

PADs and NETs in digestive system: From physiology to pathology.

Peptidylarginine deiminases (PADs) are the only enzyme class known to deiminate arginine residues into citrulline in proteins, a process known as citrullination. This is an important post-translational modification that functions in several physiological and pathological processes. Neutrophil extracellular traps (NETs) are generated by NETosis, a novel cell death in neutrophils and a double-edged sword in inflammation. Excessive activation of PADs and NETs is critically implicated in their transformation from a physiological to a pathological state. Herein, we review the physiological and pathological functions of PADs and NETs, in particular, the involvement of PAD2 and PAD4 in the digestive system, from inflammatory to oncological diseases, along with related therapeutic prospects.

Virulent and necrotrophic strategies of Bacillus thuringiensis in susceptible and resistant insects, Galleria mellonella.

Bacillus thuringiensis (Bt) is one of the most common entomopathogenic bacteria used as a biopesticide, and source of endotoxin genes for generating insect-resistant transgenic plants. The mechanisms underpinning an insect's susceptibility or resistance to B. thuringiensis are diverse. The bacterial lifecycle does not end with the death of a host, they continue to exploit the cadaver to reproduce and sporulate. Herein, we studied the progression of B. thuringiensis subsp. galleriae infection in two populations of wax moth larvae (Galleria mellonella) to gain further insight into the "arms race" between B. thuringiensis virulence and insect defences. Two doses of B. thuringiensis subsp. galleriae (spore and crystalline toxin mixtures) were administered orally to compare the responses of susceptible (S) and resistant (R) populations at ∼30% mortality each. To investigate B. thuringiensis-insect antibiosis, we used a combination of in vivo infection trials, bacterial microbiome analysis, and RNAi targeting the antibacterial peptide gloverin. Within 48 h post-inoculation, B. thuringiensis-resistant insects purged the midgut of bacteria, i.e., colony forming unit numbers fell below detectable levels. Second, B. thuringiensis rapidly modulated gene expression to initiate sporulation (linked to quorum sensing) when exposed to resistant insects in contrast to susceptible G. mellonella. We reinforce earlier findings that elevated levels of antimicrobial peptides, specifically gloverin, are found in the midgut of resistant insects, which is an evolutionary strategy to combat B. thuringiensis infection via its main portal of entry. A sub-population of highly virulent B. thuringiensis can survive the enhanced immune defences of resistant G. mellonella by disrupting the midgut microbiome and switching rapidly to a necrotrophic strategy, prior to sporulation in the cadaver.

Mucosal-Associated Invariant T Cells in the Digestive System: Defender or Destroyer?

Mucosal-associated invariant T (MAIT) cells are a subset of innate T lymphocytes that express the semi-invariant T cell receptor and recognize riboflavin metabolites via the major histocompatibility complex class I-related protein. Given the abundance of MAIT cells in the human body, their role in human diseases has been increasingly studied in recent years. MAIT cells may serve as targets for clinical therapy. Specifically, this review discusses how MAIT cells are altered in gastric, esophageal, intestinal, and hepatobiliary diseases and describes their protective or pathogenic roles. A greater understanding of MAIT cells will provide a more favorable therapeutic approach for digestive diseases in the clinical field.

The effects of temperature stress and population origin on the thermal sensitivity of Lymantria dispar L. (Lepidoptera: Erebidae) larvae.

Increased environmental temperature is one of the most frequent stresses effecting metabolic rate in herbivorous insect species. Our goal was to compare the influence of increased environmental temperature and induced thermotolerance on the activity of midgut phosphatases and brain tissue hsp70 concentration in 5th instar Lymantria dispar larvae originating from an unpolluted and polluted forest. Induced thermotolerance (larval pre-treatment at high, sub-lethal temperature) increases the species ability to overcome the negative effects of thermal stress, therefore we monitored the effect of this regime in larvae originating from both forests. Thermal regimes in this experiment predominantly influenced the alkaline phosphatases activity and it was affected by temperature, population origin, and their combined effect. Total acid phosphatases activity was changed only by the joint effect of temperature and population origin. Brain hsp70 concentration was under a significant individual and joint effect of temperature and population. In both populations, brain tissue hsp70 concentration and alkaline phosphatases activity should be taken under consideration as a battery with biomarker potential for thermal stress in L. dispar larvae as a bioindicator species.

Functional transcriptome analyses of Drosophila suzukii midgut reveal mating-dependent reproductive plasticity in females.

BACKGROUND: Insect females undergo a huge transition in energy homeostasis after mating to compensate for nutrient investment during reproduction. To manage with this shift in metabolism, mated females experience extensive morphological, behavioral and physiological changes, including increased food intake and altered digestive processes. However, the mechanisms by which the digestive system responds to mating in females remain barely characterized. Here we performed transcriptomic analysis of the main digestive organ, the midgut, to investigate how gene expression varies with female mating status in Drosophila suzukii, a destructive and invasive soft fruit pest. RESULTS: We sequenced 15,275 unique genes with an average length of 1,467 bp. In total, 652 differentially expressed genes (DEGs) were detected between virgin and mated D. suzukii female midgut libraries. The DEGs were functionally annotated utilizing the GO and KEGG pathway annotation methods. Our results showed that the major GO terms associated with the DEGs from the virgin versus mated female midgut were largely appointed to the metabolic process, response to stimulus and immune system process. We obtained a mass of protein and lipid metabolism genes which were up-regulated and carbohydrate metabolism and immune-related genes which were down-regulated at different time points after mating in female midgut by qRT-PCR. These changes in metabolism and immunity may help supply the female with the nutrients and energy required to sustain egg production. CONCLUSION: Our study characterizes the transcriptional mechanisms driven by mating in the D. suzukii female midgut. Identification and characterization of the DEGs between virgin and mated females midgut will not only be crucial to better understand molecular research related to intestine plasticity during reproduction, but may also provide abundant target genes for the development of effective and ecofriendly pest control strategies against this economically important species.

RNAi of Mannosidase-Ia in the Colorado potato beetle and changes in the midgut and peritrophic membrane.

BACKGROUND: In addition to its role in the digestive system, the peritrophic membrane (PM) provides a physical barrier protecting the intestine from abrasion and against pathogens. Because of its sensitivity to RNA interference (RNAi), the notorious pest insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata), has become a model insect for functional studies. Previously, RNAi-mediated silencing of Mannosidase-Ia (ManIa), a key enzyme in the transition from high-mannose glycan moieties to paucimannose N-glycans, was shown to disrupt the transition from larva to pupa and the metamorphosis into adult beetles. While these effects at the organismal level were interesting in a pest control context, the effects at the organ or tissue level and also immune effects have not been investigated yet. To fill this knowledge gap, we performed an analysis of the midgut and PM in ManIa-silenced insects. RESULTS: As marked phenotype, the ManIaRNAi insects, the PM pore size was found to be decreased when compared to the control GFPRNAi insects. These smaller pores are related to the observation of thinner microvilli (Mv) on the epithelial cells of the midgut of ManIaRNAi insects. A midgut and PM proteome study and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis with a selection of marker genes was performed to characterize the midgut cells and understand their response to the silencing of ManIa. In agreement with the loss of ManIa activity, an accumulation of high-mannose N-glycans was observed in the ManIa-silenced insects. As a pathogen-associated molecular pattern (PAMP), the presence of these glycan structures could trigger the activation of the immune pathways. CONCLUSION: The observed decrease in PM pore size could be a response to prevent potential pathogens to access the midgut epithelium. This hypothesis is supported by the strong increase in transcription levels of the anti-fungal peptide drosomycin-like in ManIaRNAi insects, although further research is required to elucidate this possibility. The potential immune response in the midgut and the smaller pore size in the PM shed a light on the function of the PM as a physical barrier and provide evidence for the relation between the Mv and PM. © 2022 Society of Chemical Industry.