Glycosylation in Tribolium castaneum : composition, physiological significance and exploitation for pest control

The majority of all the proteins undergoes glycosylation. This post-translational modification of proteins is involved in numerous biological processes and an erroneous glycosylation is often lethal. Following this logic interference with insect glycosylation is likely to be an effective way to control insect pests. Unfortunately, most of the knowledge on insect glycobiology comes from the research on Drosophila which lacks relevance in the context of pest insect control. This work focused on the discovery of the physiological importance of N-glycosylation in the red flour beetle, Tribolium castaneum, which is a pest and a model insect. Additionally, this PhD thesis investigates the use of glycan-binding proteins (lectins) and the disruption of N-glycosylation as control strategies against pest beetles. Lectins have high insecticidal activity against insect cells but when fed to the red flour beetle their efficiency was greatly impaired by susceptibility to proteolysis, low efficiency of passing through the peritrophic matrix and inefficient transport to the hemolymph. These factors restricting the insecticidal properties of lectins could be generalized to virtually all insecticidal proteins. Therefore these data can be used for a more rational selection of novel insecticidal toxins and enhancement of the activity of the currently used ones. By studying glycan composition, gene expression analysis and functional genomics it was determined that N-glycosylation is involved in insect metamorphosis. Regulated production of N-glycans was crucial for larval growth, progression of the life stages and development of adult appendages. Finally, disruption of the early stages of the N-glycosylation pathway appears to be promising strategy for future control of insect pests

Distribution of glycan motifs at the surface of midgut cells in the cotton leafworm (Spodoptera littoralis) demonstrated by lectin binding

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(alpha-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

Individual Osmotic Fragility Distribution: A New Parameter for Determination of the Osmotic Properties of Human Red Blood Cells

The aim of our experiments was to characterise and to validate the osmotic fragility test when applied to human blood samples with no significant alterations of osmotic fragility but with a differentiating shape of the haemolysis curve. All experiments were carried out on human erythrocytes taken from the Regional Centre of Blood Donation and Blood Therapy in Wrocław. The washed erythrocytes were exposed to near-infrared radiation (NIR) or ozonated, and the osmotic fragility test was applied. The osmotic fragility, calculated from the experimental haemolysis curve for the control and cells irradiated for 15 min, is the same within the empirical error. Calculation of the first derivative of the haemolysis curve allowed us to visualise the changes in osmotic fragility distribution after exposure to NIR. By contrast, significant changes both to the osmotic fragility value and the distribution of osmotic properties were observed after an erythrocytes ozonation procedure. Description of cell osmotic properties requires at least two parameters—the value of osmotic fragility and the slope of the haemolysis curve in the region where absorbance sharply increases due to cell haemolysis

Protein N-glycosylation and N-glycan trimming are required for postembryonic development of the pest beetle Tribolium castaneum

In holometabolous insects the transition from larva to adult requires a complete body reorganization and relies on N-glycosylated proteins. N-glycosylation is an important posttranslational modification that influences protein activity but its impact on the metamorphosis has not been studied yet. Here we used the red flour beetle, Tribolium castaneum, to perform a first comprehensive study on the involvement of the protein N-glycosylation pathway in metamorphosis. The transcript levels for genes encoding N-glycan processing enzymes increased during later developmental stages and, in turn, transition from larva to adult coincided with an enrichment of more extensively modified paucimannose glycans, including fucosylated ones. Blockage of N-glycan attachment resulted in larval mortality, while RNAi of α-glucosidases involved in early N-glycan trimming and quality control disrupted the larva to pupa transition. Additionally, simultaneous knockdown of multiple genes responsible for N-glycan processing towards paucimannose structures revealed their novel roles in pupal appendage formation and adult eclosion. Our findings revealed that, next to hormonal control, insect post-embryonic development and metamorphosis depend on protein N-glycan attachment and efficient N-glycan processing. Consequently, disruption of these processes could be an effective new approach for insect control

Toxicity, membrane binding and uptake of the Sclerotinia sclerotiorum agglutinin (SSA) in different insect cell lines

The fungal lectin purified from Sclerotinia sclerotiorum, further referred to as Sclerotinia sclerotiorum agglutinin or SSA, possesses insecticidal activity against important pest insects such as pea aphids (Acyrthosiphon pisum). This paper aims at a better understanding of its activity at cellular level. Therefore, different insect cell lines were treated with SSA. These cell lines were derived from different tissues and represent the three major orders of insects important in agriculture: CF-203 (midgut Choristoneura fumiferana, Lepidoptera), GUTAW1 (midgut, Helicoverpa zea, Lepidoptera), High5 cells (ovary, Trichoplusia ni, Lepidoptera), Sf9 (ovary cells from Spodoptera frugiperda, Lepidoptera), S2 (hemocyte, Drosophila melanogaster, Diptera), and TcA (whole body, Tribolium castaneum, Coleoptera). Although the sensitivity to SSA differs between the cell lines, SSA clearly showed toxicity in all six cell lines with median effect concentrations (EC50) ranging between 9 and 42 mu g/ml. An in-depth analysis of the mechanism of uptake in the cells revealed superior amounts of FITC-SSA at the membrane of CF-203 cells compared to Sf9 cells, while a similar small amount of SSA was internalized in both cell lines. Pre-incubation with the clathrin-mediated endocytosis inhibitor phenylarsine oxide inhibited the internalization of SSA into the CF-203 and Sf9 cells with a respective reduction of 6- and 1.7-fold. The data are discussed in relation to the importance of cellular uptake mechanism for SSA binding and cytotoxicity

Low-Level Light Therapy Protects Red Blood Cells Against Oxidative Stress and Hemolysis During Extracorporeal Circulation

Aim: An activation of non-specific inflammatory response, coagulation disorder, and blood morphotic elements damage are the main side effects of the extracorporeal circulation (ECC). Red-to-near-infrared radiation (R/NIR) is thought to be capable of stabilizing red blood cell (RBC) membrane through increasing its resistance to destructive factors. We focused on the development of a method using low-level light therapy (LLLT) in the spectral range of R/NIR which could reduce blood trauma caused by the heart-lung machine during surgery.Methods: R/NIR emitter was adjusted in terms of geometry and optics to ECC circuit. The method of extracorporeal blood photobiomodulation was tested during in vivo experiments in an animal, porcine model (1 h of ECC plus 23 h of animal observation). A total of 24 sows weighing 90–100 kg were divided into two equal groups: control one and LLLT. Blood samples were taken during the experiment to determine changes in blood morphology [RBC and white blood cell (WBC) counts, hemoglobin (Hgb)], indicators of hemolysis [plasma-free hemoglobin (PFHgb), serum bilirubin concentration, serum lactate dehydrogenase (LDH) activity], and oxidative stress markers [thiobarbituric acid reactive substances (TBARS) concentration, total antioxidant capacity (TAC)].Results: In the control group, a rapid systemic decrease in WBC count during ECC was accompanied by a significant increase in RBC membrane lipids peroxidation, while in the LLLT group the number of WBC and TBARS concentration both remained relatively constant, indicating limitation of the inflammatory process. These results were consistent with the change in the hemolysis markers like PFHgb, LDH, and serum bilirubin concentration, which were significantly reduced in LLLT group. No differences in TAC, RBC count, and Hgb concentration were detected.Conclusion: We presented the applicability of the LLLT with R/NIR radiation to blood trauma reduction during ECC