Distribution of Glycan Motifs at the Surface of Midgut Cells in the Cotton Leafworm (Spodoptera littoralis) Demonstrated by Lectin Binding

Abstract

Glycans are involved in many biological phenomena, including signal transduction, cell adhesion, immune response or differentiation. Although a few papers have reported on the role of glycans in the development and proper functioning of the insect midgut, no data are available regarding the localization of the glycan structures on the surface of the cells in the gut of insects. In this paper, we analyzed the spatial distribution of glycans present on the surface of the midgut cells in larvae of the cotton leafworm Spodoptera littoralis, an important agricultural pest insect worldwide. For this purpose, we established primary midgut cell cultures, probed these individual cells that are freely suspended in liquid medium with a selection of seven fluorescently labeled lectins covering a range of different carbohydrate binding specificities [mannose oligomers (GNA and HHA), GalNAc/Gal (RSA and SSA), GlcNAc (WGA and Nictaba) and Neu5Ac(α-2,6)Gal/GalNAc (SNA-I)], and visualized the interaction of these lectins with the different zones of the midgut cells using confocal microscopy. Our analysis focused on the typical differentiated columnar cells with a microvillar brush border at their apical side, which are dominantly present in the Lepidopteran midgut and function in food digestion and absorption, and as well as on the undifferentiated stem cells that are important for midgut development and repair. Confocal microscopy analyses showed that the GalNAc/Gal-binding lectins SSA and RSA and the terminal GlcNAc-recognizing WGA bound preferentially to the apical microvillar zone of the differentiated columnar cells as compared to the basolateral pole. The reverse result was observed for the mannose-binding lectins GNA and HHA, as well as Nictaba that binds preferentially to GlcNAc oligomers. Furthermore, differences in lectin binding to the basal and lateral zones of the cell membranes of the columnar cells were apparent. In the midgut stem cells, GNA and Nictaba bound more strongly to the membrane of these undifferentiated cells compared to the microvillar pole of the columnar cells, while SSA, HHA, WGA, and SNA-I showed stronger binding to the microvilli. Our results indicated that polarization of the midgut cells is also reflected by a specific distribution of glycans, especially between the basal and microvillar pole. The data are discussed in relation to the functioning and development of the insect midgut.

N-glycosylation Site Analysis Reveals Sex-related Differences in Protein N-glycosylation in the Rice Brown Planthopper (Nilaparvata lugens)

“…While glycoproteins carrying complex N-glycans, as enriched with RSA, play a role in transmembrane transport, and are present in lipid particles and membranes. A similar observation was made in Spodoptera midgut cells; high and oligomannose N-glycans appeared to be more abundant in the basal region of the cells, which is involved in cell-cell and cell-matrix interactions (30). On the other hand, glycans with terminal Gal/GalNAc residues were more abundant in the apical region of the columnar midgut cells, the primary site for many physiological, biological and biochemical interactions (e.g.…”

Insect midgut structures and molecules as targets of plant‐derived protease inhibitors and lectins

“…For stem cells, mannose‐binding lectins bound more strongly to the membrane, whereas N ‐acetylgalactosamine/galactose‐binding and N‐ acetylglucosamine‐binding lectins were found to be more associated with the microvilli. These data explain the differential effects of lectins with different specificities on this insect …”

Prospects, challenges and current status of RNAi through insect feeding

“…The midgut is composed of three types of epithelial cells: columnar cells (enterocyte cells) with microvilli specialized for nutrient absorption, endocrine cells, and stem cells. Presumably, is in these cells where dsRNA uptake and processing take place . There are two proposed pathways by which gut epithelium cells can internalize dsRNA from gut lumen: (i) the transmembrane channel protein‐mediated uptake pathway and (ii) the endocytic pathway.…”

“…Presumably, is in these cells where dsRNA uptake and processing take place. 16 There are two proposed pathways by which gut epithelium cells can internalize dsRNA from gut lumen: (i) the transmembrane channel protein-mediated uptake pathway and (ii) the endocytic pathway. Both systems are reported in insects and presumably they are also involved in the uptake of dsRNA from the gut and then movement into the hemolymph.…”