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The midgut, a vital component of the digestive system in arthropods, serves as an interface between ingested food and the insect’s physiology, playing a pivotal role in nutrient absorption and immune defense mechanisms. Distinct cell types, including columnar, enteroendocrine, goblet and regenerative cells, comprise the midgut in insects and contribute to its robust immune response. Enterocytes/ columnar cells, the primary absorptive cells, facilitate the immune response through enzyme secretions, while regenerative cells play a crucial role in maintaining midgut integrity by continuously replenishing damaged cells and maintaining the continuity of the immune defense. The peritrophic membrane is vital to the insect’s innate immunity, shielding the midgut from pathogens and abrasive food particles. Midgut juice, a mixture of digestive enzymes and antimicrobial factors, further contributes to the insect’s immune defense, helping the insect to combat invading pathogens and regulate the midgut microbial community. The cutting-edge single-cell transcriptomics also unveiled previously unrecognized subpopulations within the insect midgut cells and elucidated the striking similarities between the gastrointestinal tracts of insects and higher mammals. Understanding the intricate interplay between midgut cell types provides valuable insights into insect immunity. This review provides a solid foundation for unraveling the complex roles of the midgut, not only in digestion but also in immunity. Moreover, this review will discuss the novel immune strategies led by the midgut employed by insects to combat invading pathogens, ultimately contributing to the broader understanding of insect physiology and defense mechanisms. [ABSTRACT FROM AUTHOR]

The midgut of the honey bee is considered the only midgut. Processes related to digestion take place in this department. At the same time, the middle intestine takes part in the processes that indicate the immune status. The purpose of the research was to study the effect of protein feed on the morphometric parameters of the epithelium of the midgut of honeybees and the peretrophic membrane. Two groups of families participated in the study. The first group received bee pollen collected from winter rape with a protein content of 25 % as protein feed. The second group consumed dandelion meal with a protein content of 15 %. After reaching the required age, the insects were taken from the nest, and the midgut was dissected. The conducted studies indicate that the length of the enterocytes of the midgut of a honey bee at the age of 10 days ranges from 18.89 to 34.11 μm. Morphometric differences of midgut enterocytes in the experimental groups did not reveal any significant difference. It has been proven that the thickness of the pertrophic membrane in hive bees is greater than that of flying bees by 22.7 % (P < 0.001). This value depends on the area where it is placed. In particular, in the caudal part, the thickness of the pertrophic membrane is more significant on average by 25.0 % compared to the cranial part (P < 0.01). The decrease in the thickness of the midgut of flying bees is related to the nature of nutrition. Older bees consume less pollen and eat more carbohydrate feed. Along with this, it has been proven that the thickness of the pertrophic membrane depends on the composition of the diet. In hive bees that consumed protein-enriched feed, the thickness of the pertrophic membrane in the caudal part of the midgut is 22.1 % greater (P < 0.001). [ABSTRACT FROM AUTHOR]

Appendicularians play an important role in marine food webs, but the alimentary canal of hermaphroditic oikopleurids, which prevalent among urochordates, is little studied ultrastructurally. Serial microtomy revealed that the alimentary canal of Oikopleura gracilis consists of a wide pharynx and a U-shaped gut with typical for oikopleurids anatomy: oesophagus, a bilobed stomach, the vertical, short mid- and voluminous distal or rectal intestines, ending the anal papilla. The mid-intestine leaves under the right gastric lobe. TEM analysis diagnosed the following features in the alimentary canal of Oikopleura gracilis. First, the pharynx is lined with a single layer of highly flattened non-ciliated epithelium. Above the anterior part of the endostyle the pharyngeal epithelium forms a thickening – ciliated ring. It is formed by cells of two types: prismatic, ciliated microvillar cells arranged in several parallel rows, and two rows of narrow parietal cells covering the first cell type. Ciliated ring serves as a kind of first valve, prevents food reflux. Second, in the pharynx of one of the specimens three flagellate protozoans with ultrastructurally similarities of free-living kinetoplastids were found. Third, an additional row of gastric band cells was revealed in the upper part of the right gastric lobe compared to that of the gastric floor. Giant electron-lucent vesicles with homogenous content associated with rough endoplasmic reticulum found in the cytoplasm of the gastric band cell, indicating probable storage the protein digestive secrets in these cells. Large multivesicular bodies diagnosed in gastric band cells ultrastructurally corresponded to autophagosomes. Forth, a new cellular type was found, the osmiophilic enterocytes, scattered throughout the digestive epithelium, that shared ultrastructural signs with apoptotic cells. Fifth, TEM data revealed the peritrophic membrane structure and proposed its formation. The absence of its own microflora in the peritrophic membrane, i.e., the absence of an immunity barrier, may be connected with the short ontogeny of appendicularians. The ultrastructural features revealed in the alimentary canal of O. gracilis expand our knowledge of the functioning of the appendicularians and serve as the basis for further research. [ABSTRACT FROM AUTHOR]

Synthesis of chitin is a subject of great interest in the fields of physiology and immunology of crustaceans. Chitinous tissues include not only the carapace, but also an acellular membrane in the intestine called the peritrophic membrane (PM). Here, we describe the first report of chitin synthase (CHS) of a penaeid shrimp, kuruma shrimp Penaeus japonicus. Histological observations showed that fecal matter in the midgut of kuruma shrimp was wrapped with a PM, which physically separated it from the midgut epithelium. Subsequently, the chitin synthase transcript was amplified from the midgut of the shrimp. The chitin synthase gene of kuruma shrimp (MjCHS) encodes 1,523 amino acid residues. Structural prediction analysis showed that the N-terminal region of MjCHS protein included nine transmembrane helices, the middle region included the catalytic region with several conserved motifs which are found in CHSs from other arthropods, and the C-terminal region included seven transmembrane helices. Although insects have distinct exoskeletal and intestinal chitin synthases, the phylogenetic analysis suggested that crustaceans have a single CHS. MjCHS mRNA was constantly detected in the digestive tract, including the midgut and hepatopancreas of both juvenile and adult kuruma shrimp, suggesting a stable synthesis of chitin in those organs. In contrast, MjCHS mRNA was also detected in the hindgut and uropod of juvenile shrimp. After molting, the mRNA levels of MjCHS in the stomach and uropod were higher than other molting cycles. These results suggest that MjCHS contributes to chitin synthesis in both the digestive tract and the epidermis, providing fundamental insights into chitin synthesis of crustaceans. [ABSTRACT FROM AUTHOR]

Abstract Background Mosquitoes are an important vector of viral transmission, and due to the complexity of the pathogens they transmit, vector control may be the most effective strategy to control mosquito-borne diseases. Chitin is required for insect growth and development and is absent in higher animals and plants, so regulating the chitin synthesis pathway can serve as a potentially effective means to control vector insects. Most of the current research on the chitin synthase (CHS) gene is focused on chitin synthase-1 (CHS-1), while relatively little is known about chitin synthase-2 (CHS-2). Results The CHS-2 gene of Ae. albopictus is highly conserved and closely related to that of Aedes aegypti. The expression of CHS-2 in the third-instar larvae and pupal stage of Ae. albopictus was relatively high, and CHS-2 expression in adult mosquitoes reached the highest value 24 h after blood-feeding. In the fourth-instar larvae of Ae. albopictus, CHS-2 expression was significantly higher in the midgut than in the epidermis. Silencing CHS-2 in Ae. albopictus larvae had no effect on larval survival and emergence. The expression of four genes related to chitin synthesis enzymes was significantly upregulated, the expression level of three genes was unchanged, and only the expression level of GFAT was significantly downregulated. The expression of chitin metabolism-related genes was also upregulated after silencing. The level of chitin in the midgut of Ae. albopictus larvae was significantly decreased, while the chitinase activity was unchanged. The epithelium of the midgut showed vacuolization, cell invagination and partial cell rupture, and the structure of the peritrophic membrane was destroyed or even absent. Methods The expression of CHS-2 in different developmental stages and tissues of Aedes albopictus was detected by real-time fluorescence quantitative PCR (qPCR). After silencing CHS-2 of the fourth-instar larvae of Ae. albopictus by RNA interference (RNAi), the expression levels of genes related to chitin metabolism, chitin content and chitinase activity in the larvae were detected. The structure of peritrophic membrane in the midgut of the fourth-instar larvae after silencing was observed by paraffin section and hematoxylin–eosin (HE) staining. Conclusion CHS-2 can affect midgut chitin synthesis and breakdown by regulating chitin metabolic pathway-related genes and is involved in the formation of the midgut peritrophic membrane in Ae. albopictus, playing an important role in growth and development. It may be a potential target for enhancing other control methods. Graphical Abstract

The midgut, a vital component of the digestive system in arthropods, serves as an interface between ingested food and the insect’s physiology, playing a pivotal role in nutrient absorption and immune defense mechanisms. Distinct cell types, including columnar, enteroendocrine, goblet and regenerative cells, comprise the midgut in insects and contribute to its robust immune response. Enterocytes/columnar cells, the primary absorptive cells, facilitate the immune response through enzyme secretions, while regenerative cells play a crucial role in maintaining midgut integrity by continuously replenishing damaged cells and maintaining the continuity of the immune defense. The peritrophic membrane is vital to the insect’s innate immunity, shielding the midgut from pathogens and abrasive food particles. Midgut juice, a mixture of digestive enzymes and antimicrobial factors, further contributes to the insect’s immune defense, helping the insect to combat invading pathogens and regulate the midgut microbial community. The cutting-edge single-cell transcriptomics also unveiled previously unrecognized subpopulations within the insect midgut cells and elucidated the striking similarities between the gastrointestinal tracts of insects and higher mammals. Understanding the intricate interplay between midgut cell types provides valuable insights into insect immunity. This review provides a solid foundation for unraveling the complex roles of the midgut, not only in digestion but also in immunity. Moreover, this review will discuss the novel immune strategies led by the midgut employed by insects to combat invading pathogens, ultimately contributing to the broader understanding of insect physiology and defense mechanisms.

Chitin deacetylase (CDA) can accelerate the conversion of chitin to chitosan, influencing the mechanical properties and permeability of the cuticle structures and the peritrophic membrane (PM) in insects. Putative Group V CDAs SeCDA6/7/8/9 (SeCDAs) were identified and characterized from beet armyworm Spodoptera exigua larvae. The cDNAs of SeCDAs contained open reading frames of 1164 bp, 1137 bp, 1158 bp and 1152 bp, respectively. The deduced protein sequences showed that SeCDAs are synthesized as preproteins of 387, 378, 385 and 383 amino acid residues, respectively. It was revealed via spatiotemporal expression analysis that SeCDAs were more abundant in the anterior region of the midgut. The SeCDAs were down-regulated after treatment with 20-hydroxyecdysone (20E). After treatment with a juvenile hormone analog (JHA), the expression of SeCDA6 and SeCDA8 was down-regulated; in contrast, the expression of SeCDA7 and SeCDA9 was up-regulated. After silencing SeCDAV (the conserved sequences of Group V CDAs) via RNA interference (RNAi), the layer of intestinal wall cells in the midgut became more compact and more evenly distributed. The vesicles in the midgut were small and more fragmented or disappeared after SeCDAs were silenced. Additionally, the PM structure was scarce, and the chitin microfilament structure was loose and chaotic. It was indicated in all of the above results that Group V CDAs are essential for the growth and structuring of the intestinal wall cell layer in the midgut of S. exigua. Additionally, the midgut tissue and the PM structure and composition were affected by Group V CDAs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Spodoptera frugiperda is one of the world's major agricultural pests and it occurs in many countries around the world. In Lepidoptera, the peritrophic membrane in the midgut protects the midgut epithelial cells and facilitates the digestion and absorption of food. Its main components are chitin and protein. Chitin synthase (CHS), as the last enzyme in the chitin biosynthesis pathway, is very important. Here, the expression of chitin synthase B (CHSB) gene identified from midgut was inhibited by RNA interference to explore its function in the metabolism of trehalose and chitin of S. frugiperda larvae. The results were as follows: SfCHSB was highly expressed in actively feeding larvae. Second, knockdown of SfCHSB resulted in decreased expression of most genes involved in chitin metabolism, including chitinase, and abnormal phenotypes of S. frugiperda were observed. In addition, membrane-bound trehalase activity and glucose content increased, and trehalose content decreased at 24 h after dsSfCHSB injection. Trehalase activities increased significantly and the glycogen content decreased at 48 h after dsSfCHSB injection. Finally, the activity of chitinase decreased and the content of chitin increased significantly. Our results indicate that SfCHSB expression is specific, SfCHSB has a large role in regulating chitin metabolism, and it has broad application prospects in the biocontrol of S. frugiperda. [ABSTRACT FROM AUTHOR]

[Display omitted] • Ground beetles were exposed to three microplastics (MPs) polymers at the field level. • Beetle survival was unaffected, although defecation rates decreased. • Beetle digestive enzymes were altered, which were polymer and enzyme specific. • Common histological and ultrastructural alterations in midgut tissues were observed. • The existence and ecological functions of beetles may be impacted by MP pollution. Concerns about microplastic (MP) pollution in terrestrial systems are increasing. It is believed that the overall amount of MPs in the terrestrial system could be 4–23 times higher than that in the ocean. Agricultural ecosystems are among the most polluted areas with MPs. Terrestrial organisms such as ground beetles, will be more vulnerable to MPs in various agricultural soil types because they are common in garden and agricultural areas. Therefore, this work aims to assess for the first time the potential adverse effects of chronic exposure for 30 days of ground beetles to a field-realistic concentration of 2 % (w/w) of three different irregularly shaped MPs polymers: Polystyrene (PS), polyethylene terephthalate (PET), and polyamide 6 (PA; i.e., nylon 6) on their health. The results showed no effect on beetle survival; nevertheless, there was a decrease in beetle defecation rate, particularly in beetles exposed to PS-MPs, and a change in the activity of midgut digestive enzymes. The effects on digestive enzymes (amylase, protease, lipase, and α-glucosidase) were polymer and enzyme specific. Furthermore, histological and cytological studies demonstrated the decomposition of the midgut peritrophic membrane, as well as abnormally shaped nuclei, vacuolation, disordered microvilli, necrosis of goblet and columnar cells, and necrosis of mitochondria in midgut cells. Given the importance of ground beetles as predators in most agricultural and garden settings, the reported adverse impacts of MPs on their health may impact their existence and ecological functions. [ABSTRACT FROM AUTHOR]

Host plants have an effect on the susceptibility of lepidopteran insects to nucleopolyhedrovirus (NPV), but the role of the protein in peritrophic membrane (PM) of larvae has not been investigated. We determined a key PM gene (SeIIM8 gene) in Spodoptera exigua (Hübner), which plays a role in protecting against NPV infection. Then we quantified the activity of chitin synthase (SeCHS), the content of insect intestinal mucin (SeIIM) and the expression of their genes in S. exigua fed one of four foods (artificial diet, Brassica oleracea (L.), Glycine max (L.), or Ipomoea aquatica (Forsskal)) with or without inoculation of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV). Our results showed that the larval mortality was highest for NPV-infected with siRNA-injected larvae, intermediate for NPV-infected larvae, and lowest for uninfected larvae. The SeCHS activity, SeIIM content, and their relative gene expressions of S. exigua fed G. max or artificial diet were lower compared to caterpillars fed other diets. The relative SeCHS and SeIIM8 gene expression levels of larvae depended on the interaction of NPV treatment, diet, and time. This study concluded that plants affected the susceptibility of insects to NPV by altering the enzyme activity and gene expression of insects. [ABSTRACT FROM AUTHOR]

Neonicotinoids are one of the major stresses contributing to the decline in the population of honeybees. Worker bees are prone to various stress factors during foraging and are susceptible to Imidacloprid due to the reduction in the number of genes encoding for the major enzyme families responsible for the detoxification of toxins. The present study worked on the hypothesis that the dietary supplementation of Ascorbic acid (VIT C) could reduce the peroxidative damage in the worker bees of Apis cerana indica exposed to sub-lethal concentration of imidacloprid (IMD). Furthermore, we also evaluated the role and efficacy of VIT C supplementation on the cytoarchitecture of midgut tissues on exposure to IMD. Colonies of honeybees were maintained by providing sugar syrup to the control group and sugar syrup supplemented with 0.2% VIT C for the experimental group for six months. Worker bees from both groups were randomly collected and exposed to 0.001 mg/mL IMD. To study the peroxidative damage, the activities of various enzymes were analyzed. The activities of antioxidant enzymes including Catalase, Superoxide Dismutase, Glutathione S Transferase, and Glutathione Peroxidase in the hemolymph and midgut tissues of worker bees were significantly decreased due to exposure to IMD as a single agent. However, their activities showed a significant elevation under diet supplementation with VIT C. Histological examination revealed midgut tissue damage and the rupture of peritrophic membrane among the workers exposed to IMD as compared with the control group. The damage to the midgut was alleviated and the peritrophic membrane was found to be intact in the worker bees supplemented with VIT C. Our results indicated that the dietary supplementation of VIT C has the potential to maintain the redox status and thereby can offer protective potential against the peroxidative damages induced by the sub-lethal concentration of IMD.

Peritrophins are peritrophic membrane (PM) proteins that can interact with chitin fibers via chitin-binding domains. Peritrophins have essential roles in providing porosity and strength to the PM that lines the shrimp midgut. Acute hepatopancreatic necrosis disease (AHPND), caused by strains of V. parahaemolyticus, is known to initially colonize the shrimp stomach and simultaneously disrupt its structural barriers (e.g., cuticle or epithelial tissues) to reach the hepatopancreas. Although stomach and hepatopancreas were identified as target tissues involved in AHPND pathogenesis, our results indicated that peritrophin in peritrophic membrane has a crucial role in determining not only colonization of AHPND-causing bacteria but also their tissue distribution. As the interaction between Lv Peritrophin (LvPT) and WSSV (white spot syndrome virus) is not well understood, we noted that LvPT expression was upregulated in shrimp stomach challenged with either WSSV or AHPND. In an i n vitro pathogen binding assay, there was strong binding of recombinant LvPT WSSV and AHPND-causing V. parahaemolyticus , and various bacteria. Furthermore, dsRNA-mediated LvPT silencing inhibited WSSV gene expression and viral genome replication. However, downregulation of LvPT gene expression increased copies of AHPND-causing bacteria in shrimp digestive tract, and facilitated bacterial colonization in stomach. In conclusion, we speculated that LvPT might regulate bacterial colonization during AHPND, whereas in WSSV infection, LvPT silencing favored the host. Although recombinant LvPT had strong binding with WSSV, the precise role of LvPT in WSSV infection needs further investigation. These findings increased our understanding of host-pathogen interactions in AHPND and WSSV infection that can be applied in shrimp aquaculture for developing effective antibacterial and antiviral strategies. • A Peritrophin with 3 chitin-binding domains was identified from Litopenaeus vannamei. • Recombinant LvPT had strong binding ability to WSSV, and AHPND-causing V. parahaemolyticus. • Silencing LvPT in L. vannamei reduced WSSV replication. • LvPT silencing increased colonization of AHPND-causing bacteria in shrimp digestive tract. [ABSTRACT FROM AUTHOR]

Lauric acid (LA), a saturated fatty acid with 12 carbon atoms, is widely regarded as a healthy fatty acid that plays an important role in disease resistance and improving immune physiological function. The objective of this study was to determine the effects of dietary lauric acid on the growth performance, antioxidant capacity, non-specific immunity and intestinal microbiology, and evaluate the potential of lauric acids an environmentally friendly additive in swimming crab (Portunus trituberculatus) culture. A total of 192 swimming crabs with an initial body weight of 11.68 ± 0.02 g were fed six different dietary lauric acid levels, the analytical values of lauric acid were 0.09, 0.44, 0.80, 1.00, 1.53, 2.91 mg/g, respectively. There were four replicates per treatment and 8 juvenile swimming crabs per replicate. The results indicated that final weight, percent weight gain, specific growth rate, survival and feed intake were not significantly affected by dietary lauric acid levels; however, crabs fed diets with 0.80 and 1.00 mg/g lauric acid showed the lowest feed efficiency among all treatments. Proximate composition in hepatopancreas and muscle were not significantly affected by dietary lauric acid levels. The highest activities of amylase and lipase in hepatopancreas and intestine were found at crabs fed diet with 0.80 mg/g lauric acid (P < 0.05), the activity of carnitine palmityl transferase (CPT) in hepatopancreas and intestine significantly decreased with dietary lauric acid levels increasing from 0.09 to 2.91 mg/g (P < 0.05). The lowest concentration of glucose and total protein and the activity of alkaline phosphatase in hemolymph were observed at crabs fed diets with 0.80 and 1.00 mg/g lauric acid among all treatments. The activity of GSH-Px in hepatopancreas significantly increased with dietary lauric acid increasing from 0.09 to 1.53 mg/g, MDA in hepatopancreas and hemolymph was not significantly influenced by dietary lauric acid levels. The highest expression of cat and gpx in hepatopancreas were exhibited in crabs fed diet with 1.00 mg/g lauric acid, however, the expression of genes related to the inflammatory signaling pathway (relish , myd88 , traf6, nf-κB) were up-regulated in the hepatopancreas with dietary lauric acid levels increasing from 0.09 to 1.00 mg/g, moreover, the expression of genes related to intestinal inflammatory, immune and antioxidant were significantly affected by dietary lauric acid levels (P < 0.05). Crabs fed diet without lauric acid supplementation exhibited higher lipid drop area in hepatopancreas than those fed the other diets (P < 0.05). The expression of genes related to lipid catabolism was up-regulated, however, and the expression of genes related to lipid synthesis was down-regulated in the hepatopancreas of crabs fed with 0.80 mg/g lauric acid. Lauric acid improved hepatic tubular integrity, and enhanced intestinal barrier function by increasing peritrophic membrane (PM) thickness and upregulating the expression of structural factors (per44 , zo-1) and intestinal immunity-related genes. In addition, dietary 1.00 mg/g lauric acid significantly improved the microbiota composition of the intestinal, increased the abundance of Actinobacteria and Rhodobacteraceae, and decreased the abundance of Vibrio, thus maintaining the microbiota balance of the intestine. The correlation analysis showed that there was a relationship between intestinal microbiota and immune-antioxidant function. In conclusion, the dietary 1.00 mg/g lauric acid is beneficial to improve the antioxidant capacity and intestinal health of swimming crab. • Lauric acid can improve the antioxidant capacity and up-regulate the expression of anti-inflammatory factors. • Lauric acid increased the thickness of peritrophic membrane in the gut of swimming crab. • Lauric acid reduced the abundance of vibrio in the intestine. • Excessive amounts of lauric acid can lead to tissue damage and metabolic disorders. [ABSTRACT FROM AUTHOR]

Peritrophic membrane (PM) is a pellicle structure present in the midgut of some invertebrates, such as insects and crustaceans. It could isolate harmful components and pathogens in food from intestinal epithelial cells; and it also plays a role in improving digestion and absorption efficiency. So PM is important for survival of its owner. In current study, 44 PM proteins were identified in Litopenaeus vannamei by PM proteome analysis. Among these PM proteins, the Peritrophin- 44 homologous protein (LvPT44) was further studied. Chitin-binding assay indicated that LvPT44 could bind to colloidal chitin, and immunoeletron microscopy analysis shown that it was located to PM of L. vannamei. Furthermore, LvPT44 promoter was found to be activated by L. vannamei STAT and c-Jun. Besides, LvPT44 was induced by ER-stress as well as white spot syndrome virus infection. Knocked-down expression of LvPT44 by RNA inference increased the cumulative mortality of shrimp that caused by ER-stress or white spot syndrome virus. These results suggested that LvPT44 has an important role in disease resistance. [ABSTRACT FROM AUTHOR]

Chitin deacetylase (CDA) can accelerate the conversion of chitin to chitosan, influencing the mechanical properties and permeability of the cuticle structures and the peritrophic membrane (PM) in insects. Putative Group V CDAs SeCDA6/7/8/9 (SeCDAs) were identified and characterized from beet armyworm Spodoptera exigua larvae. The cDNAs of SeCDAs contained open reading frames of 1164 bp, 1137 bp, 1158 bp and 1152 bp, respectively. The deduced protein sequences showed that SeCDAs are synthesized as preproteins of 387, 378, 385 and 383 amino acid residues, respectively. It was revealed via spatiotemporal expression analysis that SeCDAs were more abundant in the anterior region of the midgut. The SeCDAs were down-regulated after treatment with 20-hydroxyecdysone (20E). After treatment with a juvenile hormone analog (JHA), the expression of SeCDA6 and SeCDA8 was down-regulated; in contrast, the expression of SeCDA7 and SeCDA9 was up-regulated. After silencing SeCDAV (the conserved sequences of Group V CDAs) via RNA interference (RNAi), the layer of intestinal wall cells in the midgut became more compact and more evenly distributed. The vesicles in the midgut were small and more fragmented or disappeared after SeCDAs were silenced. Additionally, the PM structure was scarce, and the chitin microfilament structure was loose and chaotic. It was indicated in all of the above results that Group V CDAs are essential for the growth and structuring of the intestinal wall cell layer in the midgut of S. exigua. Additionally, the midgut tissue and the PM structure and composition were affected by Group V CDAs.

Onchocerciasis is a neglected tropical disease (NTD) caused by Onchocerca Diesing 1841 (Spirurida: Onchocercidae) nematodes transmitted by blackflies. It is associated with poverty and imposes a significant health, welfare and economic burden on many tropical countries. Current methods to visualize infections within the vectors rely on invasive methods. However, using micro‐computed tomography techniques, without interference from physical tissue manipulation, we visualized in three dimensions for the first time an L1 larva of an Onchocerca species within the thoracic musculature of a blackfly, Simulium damnosum s.l. Theobald 1903 (Diptera: Simuliidae), naturally infected in Ghana. The possibility that thicker peritrophic membranes in savannah flies could account for their lower parasite loads was not supported, but there were limits to our analysis. While there were no statistically significant differences between the mean thicknesses of the peritrophic membranes, in the anterior, dorsal and ventral regions, of forest and savannah blackflies killed 34–48 min after a blood‐meal, the thickness of the peritrophic membrane in the posterior region could not be measured. Micro‐computed tomography has the potential to provide novel information on many other parasite/vector systems and impactful images for public engagement in health education. [ABSTRACT FROM AUTHOR]

Spodoptera frugiperda is one of the world’s major agricultural pests and it occurs in many countries around the world. In Lepidoptera, the peritrophic membrane in the midgut protects the midgut epithelial cells and facilitates the digestion and absorption of food. Its main components are chitin and protein. Chitin synthase (CHS), as the last enzyme in the chitin biosynthesis pathway, is very important. Here, the expression of chitin synthase B (CHSB) gene identified from midgut was inhibited by RNA interference to explore its function in the metabolism of trehalose and chitin of S. frugiperda larvae. The results were as follows: SfCHSB was highly expressed in actively feeding larvae. Second, knockdown of SfCHSB resulted in decreased expression of most genes involved in chitin metabolism, including chitinase, and abnormal phenotypes of S. frugiperda were observed. In addition, membrane-bound trehalase activity and glucose content increased, and trehalose content decreased at 24 h after dsSfCHSB injection. Trehalase activities increased significantly and the glycogen content decreased at 48 h after dsSfCHSB injection. Finally, the activity of chitinase decreased and the content of chitin increased significantly. Our results indicate that SfCHSB expression is specific, SfCHSB has a large role in regulating chitin metabolism, and it has broad application prospects in the biocontrol of S. frugiperda.

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. [ABSTRACT FROM AUTHOR]

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BACKGROUND The insect‐pathogenic bacterium Yersinia entomophaga MH96 is currently under development as a microbial pesticide active against various pasture and crop pests such as the diamondback moth Plutella xylostella and the cotton bollworm Helicoverpa armigeria. To enable nonrestricted field trials of Y. entomophaga MH96, information on the persistence and nontarget effects of the bacterium and its Yen‐Tc proteinaceous toxin are required. RESULTS: The Y. entomophaga Yen‐Tc associated toxin was found to have limited persistence on foliage and is inactivated by UV light. The Yen‐Tc was rapidly degraded in ovine or bovine rumen fluid or the intestinal fluid of H. armigera. In H. armigera an intestinal protein of >50 kDa was found to cleave the Yen‐Tc bond. Assessment of Y. entomophaga persistence on foliage and in soil found that after 42 days the bacterium could not be detected in soil at 20% soil moisture content but persisted for 72 days at 30–40% soil moisture. Nontarget effects of Y. entomophaga towards earthworms found that the bacterium afforded no adverse effects on worm growth or behavior. A summary of historic Yen‐Tc and Y. entomophaga persistence and toxicity data is presented. CONCLUSION: The bacterium Y. entomophaga and its Yen‐Tc associated toxin have limited persistence in the environment, with the Yen‐Tc being susceptible to UV inactivation and proteolytic degradation, and the bacterium persisting longer in soil of a high moisture content. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract Background Mosquitoes transmit many vector-borne infectious diseases including malaria, dengue, chikungunya, yellow fever, filariasis, and Japanese encephalitis. The insecticidal δ-endotoxins Cry4, Cry11, and Cyt produced from Bacillus thuringiensis have been used for bio-control of mosquito larvae. Cry δ-endotoxins are synthesised as inactive protoxins in the form of crystalline inclusions in which they are processed to active toxins in larval midgut lumen. Previously, we demonstrated that the activated Cry4Ba toxin has to alter the permeability of the peritrophic membrane (PM), allowing toxin passage across PM to reach specific receptors on microvilli of larval midgut epithelial cells, where the toxin undergoes conformational changes, followed by membrane insertion and pore formation, resulting in larval death. A peritrophic membrane (PM)-binding calcofluor has been proposed to inhibit chitin formation and enhance baculovirus infection of lepidopteran Trichoplusia ni. Methods In this study, Aedes aegypti larvae were fed with the calcofluor and Cry4Ba toxin to investigate the effect of this agent on the toxicity of the Cry4Ba toxin. Results Calcofluor displayed an enhancing effect when co-fed with the Cry4Ba wild-type toxin. The agent could restore the killing activity of the partially active Cry4Ba mutant E417A/Y455A toward Ae. aegypti larvae. PM destruction was observed after larval challenge with calcofluor together with the toxin. Interestingly, calcofluor increased Cry4Ba toxin susceptibility toward semi-susceptible Culex quinquefasciatus larvae. However, calcofluor alone or in combination with the toxin showed no mortality effect on non-susceptible fresh-water fleas, Moina macrocopa. Conclusions Our results suggest that PM may contribute to the resistance of the mosquito larvae to Cry4Ba toxin. The PM-permeability alternating calcofluor might be a promising candidate for enhancing insect susceptibility, which will consequently improve Cry4Ba efficacy in field settings in the future.

Introduction and purpose: Leishmaniasis are neglected diseases that are endemic in more than half of the provinces of Iran. The causative agent of this disease is Leishmania protozoa and its transmission to human is through the bites of phlebotomine sand flies. This study reviews the growth and development of Leishmania in the body of sand flies at the molecular level. Methods: In this study a narrative review was carried out. The corpus was collected by searching the keywords of Leishmania, sand fly, phlebotomus, lutzomyia in the databases of PubMed, Scopus, SID, Web of Science, Ovid Medline, and Google Scholar search engine. The studied articles were those which has been published on the interaction between the sand fly vectors and Leishmania parasite until 2018. Results: The parasites grow when they encounter natural barriers in the body of the sand flies, including proteolytic enzymes, peritrophic membrane, excretion of the digestive system, and immune system in sand fly vectors. Attachment of Leishmania to sand fly digestive tract determines the ability of sand fly to support the development of parasite, which leads to the transmission of leishmaniasis.Conclusion: Studies on the development and life cycle of Leishmania within the body of sand flies are of utmost importance due to the spread of leishmaniasis in Iran and the world, difficulty in their control, and lack of effective vaccine products against leishmaniasis. The findings of the current study aimed at identifying and enhancing the natural barriers to the development of Leishmania in sand fly vectors and introducing new strategies for the control of leishmaniasis.

Butyrate is a fermentation byproduct of gut microbiota and is susceptible to chronic oxidative stress. This study investigates the mitigative effects of sodium butyrate (SBT) on growth inhibition and intestinal damage induced by glycinin in juvenile Chinese mitten crab (Eriocheir sinensis). All four experimental diets containing 80 g/kg glycinin were formulated with 0, 10, 20 and 40 g/kg SBT respectively. There was no glycinin or SBT in the control diet. Juvenile crabs (0.33 ± 0.01g) were respectively fed with these five diets for eight weeks. The diets with 10 and 20 g/kg SBT significantly improved the survival and weight gain of the crabs compared with those in the 0 g/kg SBT group, and showed no difference with the control group. The crabs fed diets containing glycinin without SBT had lower glutathione and glutathione peroxidase activities but higher malondialdehyde in the intestine than those in the control group. Moreover, dietary glycinin decreased the lysozyme and phenoloxidase activities and improved the level of histamine in the intestine compared with the control group, while the supplementation of SBT counteracted these negative effects. The addition of SBT could also restore the impaired immunity and morphological structure of the intestine. Dietary SBT could increase the mRNA expression of antimicrobial peptides genes (anti-lipopolysaccharide factor 1 and 2) and decrease the content of pro-inflammatory factor TNF-α. The SBT could restore the intestinal microbial community disorganized by glycinin. The abundance of pathogenic bacteria (Aeromonas , Vibrio and Pseudomonas) decreased significantly and the potential probiotic bacteria (Bacillus , Lactobacillus , Chitinibacter and Dysgonomonas) increased significantly in the 10 g/kg SBT group. This study suggests that sodium butyrate supplementation can mitigate the negative effects induced by glycinin such as growth inhibition, intestinal inflammation and reduction of beneficial flora in the gut. • The SBT supplement in diet ameliorate the growth inhibition induced by glycinin in E. sinensis. • Dietary SBT restored the impaired immunity and morphological structure of the intestine in E. sinensis. • Dietary SBT reshaped the community of gut microbiota induced by glycinin in crab diets. • The supplementation of 1–2% SBT is recommended when the level of glycinin is over 80 g/kg in the diet of E. sinensis. [ABSTRACT FROM AUTHOR]

The aim of this current study is to focus on the histopathological changes that happens to the midgut of the cotton leaf worm Spodoptera littoralis (Boisd) larvae especially in the Peritrophic membrane as a main defense line against the occulosion bodies OBs (A crystalline protein matrix which occludes the nucleocapsids) of the Spodoptera littoralis nucleopolyhedrovirus from a local Iraqi isolation that was used as a biological control factor to control this pest. The high specialty of the baculovirus when used as a biological control factor for certain insect families has an important role in full control programs to control pests, especially the SpoNPV virus which is highly specialized in infecting the cotton leaf worm S. littoralis larvae. And it was pointed out by results that there was dissociation and disintegration in the midgut Peritrophic membrane of the larva of the cotton leaf worm at the forth larva stage after 24 hours of exposing them to semi-synthesized nutrition with OBs of the SpoNPV. The microscopic test of the cross section of the larva's midgut showed that after 72 hours of viral infection virus particles have speeded through the rest of the inner tissue of the digestive system and invaded it to reach the fat bodies and tracheal system up to the dermis tissues and the outer cuticle cells of the larva. According to the results of the histopathological study of the infected S. littoralis with the SpoNPV, the virus is considered to be an effective, specialized and ecological safe biological control factor to control this serious pest.

ODV-E66 is a major envelope proteins of baculovirus occlusion derived virus (ODV) with chondroitinase activity. Here, we studied the roles of ODV-E66 during Helicoverpa armigera nucleopolyhedrovirus (HearNPV) primary infection. ODV-E66 is a late viral protein dispensable for BV production and ODV morphogenesis. Deletion of odv-e6 6 had a profound effect on HearNPV oral infectivity in 4th instar larvae with a 50% lethal concentration (LC 50) value of 26 fold higher than that of the repaired virus, compared to in 3rd instar larvae. Calcofluor white, an agent which destroys the peritrophic membrane (PM), could rescue the oral infectivity of odv-e66 deleted HearNPV, implying the PM may be the target of ODV-E66. In vitro assays showed HearNPV ODV-E66 has chondroitinase activity. Electron microscopy demonstrated that odv-e66 deletion alleviated the damage to the PM caused by HearNPV infection. These data suggest an important role of ODV-E66 in the penetration of the PM during oral infection. [ABSTRACT FROM AUTHOR]

Chitin, poly (β-(1→4)-N-acetyl-d-glucosamine), is an important biopolymer for insects that is utilized as a major component of peritrophic membrane. The chitin content in peritrophic membrane is of expedient interest from a pest control perspective, although it is hard to quantify chitin. In this study, we establish a facile method for the quantification of chitin in peritrophic membrane by image processing. In this method, chitin was indirectly quantified using chitosan–I3− complex, which exhibited a specific red-purple color. A calibration curve using a chitosan solution showed good linearity in a concentration range of 0.05–0.5 μg/μL. We quantified the amount of chitin in peritrophic membrane of Spodoptera litura (Lepidoptera: Noctuidae) larvae using this method. Throughout the study, only common inexpensive regents and easily attainable apparatuses were employed. This method can be easily applied to the sensitive quantification of the amounts of chitin and chitosan in materials by wide range of researchers. Abbreviations: LOD: limit of detection; LOQ: limit of quantification; ROI: region of interest; RSD: relative standard deviation. Chitin in peritrophic membrane of insects was quantified by image processing with an easy, inexpensive and sensitive method. [ABSTRACT FROM AUTHOR]

• First report on the N -glycome of the peritrophic matrix of Colorado potato beetle. • 97% of the N -glycan pool in the PM consists of three dominating groups. • These groups are paucimannoses, mono-fucosylated paucimannoses and high mannoses. • The three most abundant N -glycans are GlcNAc2Man3Fuc, GlcNAc2Man3 and GlcNAc2Man5. • NGRGs are identified in the tissue specific transcriptome of the midgut epithelium. • The N -glycome of the PM correlates with the presence of the NGRGs in the epithelium. The insect peritrophic membrane (PM) is a non-cellular structure composed of secreted proteins imbedded in a proteoglycan matrix together with chitin. It separates the midgut epithelium from the intestinal contents, and functions in the digestion of food. Furthermore it acts as a protective barrier against abrasive particles and microbial infections. Here we studied for the first time the N -glycome of the PM. We identified the N -glycan structures present in the PM of the Colorado potato beetle (CPB) at the fourth larval stage using MALDI-TOF mass spectrometry. In parallel, we correlated the N -glycan data to the presence of the N -glycosylation related genes (NGRGs) in the transcriptome of epithelial midgut cells. The presumed activities of the identified genes support the N -glycan profile resolved for the proteins in the PM. To our knowledge these data are the first report on the N -glycome of the PM of a pest insect. These results will contribute to the study of the importance of N -glycosylation in the function and structure of the PM. In addition, the data can help to find novel targets and design better biopesticides for pest control. [ABSTRACT FROM AUTHOR]

The Cydia pomonella granulovirus (CpGV) GP37 has synergistic effects on the infectivity of nucleopolyhedroviruses (NPVs), however, the mechanism employed is unclear. In this study, in vitro and in vivo binding assays indicated that GP37 efficiently bound to the midgut peritrophic membrane (PM) of Spodoptera exigua larvae. Treatment with GP37 led to the damage of the PM’s compacted structure and the generation of the PM perforations, and the enhancement of the PM’s permeability. qPCR results further demonstrated that GP37 increased the ability of occlusion-derived virions (ODV) to cross the PM. R18-labeling experiments exhibited that GP37 also promoted the fusion of ODVs and insect midgut epithelia. Altogether, our present results revealed that the synergistic mechanism of GP37 to the infectivity of NPV might involve two parts. GP37 damaged the integrity of the PM after binding, which enhanced the PM’s permeability and increased the ability of ODVs to cross the PM, finally facilitating the ODVs reaching the midgut. In addition, GP37 promoted the fusion of ODVs and insect midgut epithelia. Our data expand the understanding of the mechanism used by baculovirus synergistic factors and provide a foundation for the development of high-efficiency baculoviral insecticides. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Three silkworm midgut chitin deacetylases are cloned, expressed and purified. • Enzymatic activities of the chitin deacetylases toward peritrophic membrane are provided. • Roles of the chitin deacetylases in peritrophic membrane function are proposed. Abstract Peritrophic membrane (PM) is a chitin and protein-containing extracellular matrix that lines the midgut in most insect species, functioning as a barrier to exogenous toxins and pathogens. Midgut chitin deacetylases (CDAs) are chitin-modifying enzymes known to alter the mechanical property and permeability of PM. However, biochemical properties and specific roles of these enzymes remain elusive. In this study, the midgut-expressed CDAs (Bm CDA6, Bm CDA7 and Bm CDA8) from Bombyx mori were cloned, recombinantly expressed and purified and their enzymatic activities toward PM chitin were determined. Of the three enzymes, Bm CDA7 exhibited the highest activity (0.284 μmol/min/μmol), while Bm CDA8 showed lower activity of 0.061 μmol/min/μmol. Bm CDA6 was inactive towards PM chitin. Gene expression patterns indicated that although all three CDA genes were specifically expressed in the anterior midgut, they differed in their temporal expression patterns. BmCDA6 was expressed almost exclusively at the mid-molt stage, the stage when the PM was thick and with multiple chitin layers. Unlike BmCDA6 , high expression levels of BmCDA7 and BmCDA8 were observed only at the feeding stage, the stage when the PM is thin and with fewer chitin layers. The different gene expression patterns and biochemical characteristics provide new information about the functional specialization among Bm CDA6, Bm CDA7 and Bm CDA8 proteins. [ABSTRACT FROM AUTHOR]

The present study aims to assess the anti‐nutritional effect of pectin and to better understand rapeseed meal (RSM) as a feed ingredient for Chinese mitten crab, Eriocheir sinensis. A semipurified diet containing 280 g/kg fish meal was formulated as a control diet (DF). A second diet (DP) was supplemented with 26.7 g/kg pectin; the amount was estimated to be equal to that in the third diet (DR), which was formulated to include 400 g/kg RSM. Another diet (DRPase) was supplemented with 12 g/kg pectinase based on DR in which pectin was degraded by pectinase. The diets were randomly fed to twelve tanks of the crab for 8 weeks. Compared with the crabs fed DF, the specific growth rate, protein efficiency ratio, apparent digestibility coefficients (ADC) of dry matter and protein, activity of aspartate transaminase (AST) and trypsin (TP), and high‐density lipoprotein (HDL) in the serum decreased significantly in the crabs fed DR; the ADC of dry matter, activity of AST and serum HDL decreased significantly in the crabs fed Dp, but they were higher than in the crabs fed DR. Compared with the crabs fed DR, the ADC of dry matter, activity of AST and TP, and serum HDL increased significantly in the crabs fed DRPase. The mRNA expression activity of both Es‐pl44 and Es‐pp1 was DF < DP < DRPase < DR. These results indicate that pectin contributed to low utilization of RSM in Chinese mitten crab. [ABSTRACT FROM AUTHOR]

Abstract This study investigates the effects of two soybean antigens (glycinin and β-conglycinin) as an antinutritional substance in the diet on the growth, digestive ability, intestinal health and microbiota of juvenile Chinese mitten crabs (Eriocheir sinensis). The isonitrogenous and isolipidic diets contained two soybean antigens at two levels each (70 and 140 g/kg β-conglycinin, 80 and 160 g/kg glycinin) and a control diet without β-conglycinin or glycinin supplementation, and were used respectively to feed juvenile E. sinensis for seven weeks. Dietary inclusion of either glycinin or β-conglycinin significantly reduced crab survival and weight gain. The crabs fed diets containing soybean antigens had higher malondialdehyde concentrations and lower catalase activities in the intestine than those in the control. The activities of trypsin and amylase in the intestine were suppressed by dietary β-conglycinin and glycinin. Dietary glycinin or β-conglycinin impaired the immunity and morphological structure of intestine, especially the peritrophic membrane. The mRNA expression of constitutive and inducible immune responsive genes (lipopolysaccharide-induced TNF-α factor and interleukin-2 enhancer-binding factor 2) increased while the mRNA expression of the main genes related to the structural integrity peritrophic membrane (peritrophin-like gene and peritrophic 2) significantly decreased in the groups with soybean antigen addition. Soybean antigen could also change the intestinal microbial community. The abundance of pathogenic bacteria (Ochrobactrum , Burkholderia and Pseudomonas) increased significantly in both soybean antigen groups. Although pathogenic bacteria Vibrio were up-regulated in the glycinin group, the abundance of Dysgonomonas that degraded lignocellulose and ameliorated the gut environment decreased in the glycinin group. This study indicates that existence of soybean antigens (glycinin or β-conglycinin) could induce gut inflammation, reshape the community of gut microbiota, and cause digestive dysfunction, ultimately leading to impaired growth in crabs. Highlights • Soybean derived glycinin or β-conglycinin significantly reduced survival and weight gain of E. sinensis. • β-conglycinin and glycinin could induce inflammation and gut microbial community shift of E. sinensis. • Dietary glycinin can pose more seriously adverse effects than β-conglycinin in E. sinensis. [ABSTRACT FROM AUTHOR]

New approaches have led to advances in the molecular knowledge of digestion and nutrient absorption in insects. Cell fractionation with proteomics revealed the molecular composition of the insect midgut microvill and associated with RNA interference supported a model of microapocrine secretion. The use of mainly transcriptomics confirmed the widespread occurrence of a midgut mucus layer and clarified the differences between peritrophic membrane peritrophins and cuticular proteins analogous to peritrophins. The use of recombinant enzymes and bioinformatic tools resulted in a better understanding of lysosomal enzymes as digestive enzymes in curculionidae beetles and hemipterans, the characterization of several plant cell wall-degrading enzymes and the finding of their origin by horizontal gene transference from bacteria. Proteomics, transcriptomics and in vivo experiments with inhibitors clarified aspects of midgut buffering, nutrient absorption and midgut fluid fluxes. In the last case, the results support a model of digestive enzyme recycling to avoid enzyme excretion. [ABSTRACT FROM AUTHOR]

Midgut cell and peritrophic membranes of grain pest Agrotis segetum larvae were examined by light microscope. Six biotinylated lectins were used in the assays by applying histochemical methods (Avidin-Biotin-Peroxidase). The aim of this study was to find lectins binding on peritrophic membranes if they were available as insecticidal agents. Results of the study indicated that lectins BPA (Bauhinia purpurea) and GS-I (Griffonia simplicifolia) strongly stained whereas, PNA (Pea Nut Agglutinin) and UEA-1 (Ulex europaeus) moderately stained the tissues. However, WGA (Wheat Germ Agglutinin) and Con-A (Canavalia ensiformis) stained the tissues weakly. The common feature of two binding proteins, BPA and GS-I lectin was binding to D- galactose. Our examinations revealed that D-galactose mostly exists in A. segetum larvae midgut cell membranes and peritrophic membrane and this means BPA and GSI lectins can be used as insecticidal agents against A. segetum. [ABSTRACT FROM AUTHOR]

The insect midgut secretes a semi-permeable, acellular peritrophic membrane (PM) that maintains intestinal structure, promotes digestion, and protects the midgut from food particles and pathogenic microorganisms. Peritrophin is an important PM protein (PMP) in the PM. Here, we identified 11 peritrophins with 1–16 chitin binding domains (CBDs) comprising 50–56 amino acid residues. Multiple CBDs in the same peritrophin clustered together, rather than by species. The CBD contained six highly conserved cysteine residues, with the key feature of amino acids between them being CX11-15CX5CX9-14CX11-12CX6-7C. Peritrophins with 2 and 4 CBDs (Bm09641 and Bm01504, respectively), and with 1, 8, and 16 CBDs (Bm11851, Bm00185, and Bm01491, respectively) were mainly expressed in the anterior midgut, and throughout the midgut, respectively. Survival rates of transgenic silkworms with Bm01504 overexpression (Bm01504-OE) and knockout (Bm01504-KO) infected with B. morinucleopolyhedrovirus (BmNPV) were significantly higher and lower, whereas expression of the key viral gene, p10, were lower and higher, respectively, compared with wild type (WT). Therefore, Bm01504-OE and Bm01504-KO transgenic silkworms were more and less resistant, respectively, to BmNPV. Bm01504 plays important roles in resisting BmNPV invasion. We provide a new perspective for studying PM function, and reveal how the silkworm midgut resists invasive exogenous pathogenic microorganisms.

Serine peptidases (SPs) are responsible for most primary protein digestion in Lepidoptera species. An expansion of the number of genes encoding trypsin and chymotrypsin enzymes and the ability to upregulate the expression of some of these genes in response to peptidase inhibitor (PI) ingestion have been associated with the adaptation of Noctuidae moths to herbivory. To investigate whether these gene family expansion events are common to other Lepidoptera groups, we searched for all genes encoding putative trypsin and chymotrypsin enzymes in 23 publicly available genomes from this taxon. Phylogenetic analysis showed that several gene family expansion events may have occurred in the taxon’s evolutionary history and that these events gave rise to a very diverse group of enzymes, including proteins lacking the canonical SP catalytic triad. The expression profile of these enzymes along the midgut and the secretion mechanisms by which these enzymes enter the luminal content were also analyzed in Spodoptera frugiperda larvae using RNA-seq and proteomics. These results support the proposal of a midgut countercurrent flux responsible for the direction of these proteins to the anterior portion of the midgut and show that these enzymes reach the midgut lumen via both exocytosis and microapocrine secretion mechanisms.

The mid gut in insects is usually lined with a peritrophic membrane (PM), which protects it from microbial infections and physical contact with ingested food. Calcofluor is an optical brightener that has strong chitin-binding properties, which disrupt the chitin-protein structure of PM. In this study, we investigated the effects of calcofluor on the structure of the PM, activity of some common digestive enzymes and biological characteristics of the Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae). Twenty hours after feeding on a calcofluor-containing diet, the PM was completely eliminated. We recorded significant effects of calcofluor on the activity of digestive enzymes in the mid and hindguts of larvae. In both these regions of the gut, in particular, there were significant increases in the activity of α-amylase, α-galactosidase and trypsin. The activities of α-glucosidase and β-glucosidase in the midgut of treated larvae were lower than in the control, but were significantly higher in the hindgut of treated larvae. The larvae that fed on on calcofluor for 20 h, took longer to complete their larval and pupal stages, were lighter in weight and fewer reached maturity and laid fewer eggs. Results of this and previous studies highlight the crucial role of PM in enzyme recycling and digestion. PM disruption may negatively affect insect development on the one hand, by decreasing the efficiency of food digestion and, on the other, by increasing the costs associated with an increased enzyme demand and disrupted enzyme recycling mechanism.