No abstract

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.

No 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.

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 (EC) ranging between 9 and 42 μg/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.