N-glycosylation site analysis reveals sex-related differences in protein N-glycosylation in the rice brown planthopper (Nilaparvata lugens)

Glycosylation is a common modification of proteins and critical for a wide range of biological processes. Differences in protein glycosylation between sexes have already been observed in humans, nematodes and trematodes, and have recently also been reported in the rice pest insect Nilaparvata lugens. Although protein N-glycosylation in insects is nowadays of high interest because of its potential for exploitation in pest control strategies, the functionality of differential N-glycosylation between sexes is yet unknown. In this study, therefore, the occurrence and role of sex-related protein N-glycosylation in insects were examined. A comprehensive investigation of the N-glycosylation sites from the adult stages of N. lugens was conducted, allowing a qualitative and quantitative comparison between sexes at the glycopeptide level. N-glycopeptide enrichment via lectin capturing using the high mannose/paucimannose-binding lectin Concanavalin A, or the Rhizoctonia solani agglutinin which interacts with complex N-glycans, resulted in the identification of over 1300 N-glycosylation sites derived from over 600 glycoproteins. Comparison of these N-glycopeptides revealed striking differences in protein N-glycosylation between sexes. Male- and female-specific N-glycosylation sites were identified, and some of these sex-specific N-glycosylation sites were shown to be derived from proteins with a putative role in insect reproduction. In addition, differential glycan composition between males and females was observed for proteins shared across sexes. Both lectin blotting experiments as well as transcript expression analyses with complete insects and insect tissues confirmed the observed differences in N-glycosylation of proteins between sexes. In conclusion, this study provides further evidence for protein N-glycosylation to be sex-related in insects. Furthermore, original data on N-glycosylation sites of N. lugens adults are presented, providing novel insights into planthopper's biology and information for future biological pest control strategies

Flexibility and extracellular opening determine the interaction between ligands and insect sulfakinin receptors

Despite their fundamental importance for growth, the mechanisms that regulate food intake are poorly understood. Our previous work demonstrated that insect sulfakinin (SK) signaling is involved in inhibiting feeding in an important model and pest insect, the red flour beetle Tribolium castaneum. Because the interaction of SK peptide and SK receptors (SKR) initiates the SK signaling, we have special interest on the structural factors that influence the SK-SKR interaction. First, the three-dimensional structures of the two T. castaneum SKRs (TcSKR1 and TcSKR2) were generated from molecular modeling and they displayed significance in terms of the outer opening of the cavity and protein flexibility. TcSKR1 contained a larger outer opening of the cavity than that in TcSKR2, which allows ligands a deep access into the cavity through cell membrane. Second, normal mode analysis revealed that TcSKR1 was more flexible than TcSKR2 during receptor-ligand interaction. Third, the sulfated SK (sSK) and sSK-related peptides were more potent than the nonsulfated SK, suggesting the importance of the sulfate moiety

Protein N-glycosylation in the hemipteran pest insect Nilaparvata lugens

N-glycosylation is one of the most abundant and conserved protein modifications in eukaryotes. In insects, protein N-glycosylation plays a vital role in growth, development and survival, which makes it an interesting target for future pest control strategies. So far, all research on protein N-glycosylation has been conducted in holometabolous (pest) insects, while there is no information available for hemimetabolous insects. To fill this gap in our knowledge, this thesis focuses on protein N-glycosylation in the hemimetabolous model insect Nilaparvata lugens. N. lugens, or the brown planthopper, is one of the most notorious pest insects of cultured rice, and outbreaks of this insect lead to enormous economic losses each year. When studying protein N-glycosylation in N. lugens, no dynamics were observed during post-embryonic development. In contrast, discrepancies in the N-glycomes of adult morphs were observed between adult sexes, rather than between wing types. The female adult develops a unique N-glycan fingerprint, with a distinct increase in high mannose N-glycans. In addition, enrichment of glycopeptides revealed both differential glycosylation between males and females of proteins occurring in both sexes, as well as sex-specific glycoproteins. All together these data provide novel insights into planthopper’s biology, confirm that protein N-glycosylation is sex-related in this insect, and offer novel targets for the control of N. lugens.N-glycosylatie is één van de meest voorkomende en geconserveerde modificaties van eiwitten in eukaryoten. In insecten speelt dit proces een cruciale rol in hun groei, ontwikkeling en overleving, wat inhoudt dat dit proces een interessant doelwit kan zijn voor toekomstige strategieën voor het bestrijden van insecten. Onderzoek naar eiwit N-glycosylatie in insecten werd tot dusver enkel uitgevoerd in (plaag)insecten die een complete metamorfose ondergaan tijdens hun ontwikkeling, terwijl er geen informatie beschikbaar is voor hemimetabole insecten ofwel insecten die een incomplete gedaanteverwisseling ondergaan. Om dit gebrek aan informatie aan te vullen, focust deze thesis zich op eiwit N-glycosylatie in het hemimetabool plaaginsect Nilaparvata lugens. N. lugens, of de bruine dwergcicade, is één van de meest schadelijke plaaginsecten van rijstplanten, en een uitbraak van dit insect kan leiden tot enorme economische verliezen. Tijdens het bestuderen van eiwit N-glycosylatie in N. lugens werd duidelijk dat er geen verschillen waarneembaar zijn tijdens de post-embryonale ontwikkeling. Echter, verschillen in N-glycomen werden geobserveerd voor adulte insecten, en traden op tussen mannelijke en vrouwelijke adulten, en niet tussen de vleugelvormen. Het vrouwelijke volwassen insect ontwikkelt een uniek N-glycaan profiel met een duidelijke toename in hoog mannose N-glycanen. Verder bleek ook na verrijking van glycopeptiden dat zowel verschillen in glycosylatie tussen mannelijke en vrouwelijke insecten voorkomen voor eiwitten aanwezig in beide geslachten, alsook dat geslachtsspecifieke glyco-eiwitten voorkomen. Samengevat, deze resultaten bieden nieuwe inzichten in de biologie van cicaden, bevestigen dat eiwit N-glycosylatie geslachtsafhankelijk is in dit insect, en bieden nieuwe doelwitten voor toekomstige bestrijding van N. lugens

Let's talk about sexes : sex-related N-glycosylation in ecologically important invertebrates

Parasitic helminths and pest insects are organisms with great ecological importance, having direct or indirect detrimental effects on people's lives worldwide. Several reports in literature indicate that the glycan repertoire of parasites plays important roles in host-parasite interactions and modulation and evasion of the host immune system, while insect glycans are essential for their survival, growth and development. Although glycosylation is the result of a highly conserved machinery, differences between species and between different stages of one organism's life cycle occur. This review provides insight into recent glycomics studies both for helminths and insects, focussing on sex differences and the role of carbohydrate structures in reproduction. Information on the differential N-glycosylation process between males and females can generate a better understanding of the biology and physiology of these economic important organisms, and can contribute to the discovery of novel anti-fecundity vaccine candidates and drug targets, as well as in the elaboration of innovative pest management strategies

Protection of rice against Nilaparvata lugens by direct toxicity of sodium selenate

Nilaparvata lugens is one of the most notorious pest insects of cultured rice, and outbreaks of N. lugens cause high economic losses each year. While pest control by chemical pesticides is still the standard procedure for treating N. lugens infections, excessive use of these insecticides has led to the emergence of resistant strains and high pesticide residues in plants for human consumption and the environment. Therefore, novel and environment-friendly pest control strategies are needed. In previous studies, selenium was shown to protect selenium-accumulating plants from biotic stress. However, studies on nonaccumulator (crop) plants are lacking. In this study, rice plants (Oryza sativa, Nipponbare) were treated with sodium selenate by seed priming and foliar spray and then infested with N. lugens. Brown planthoppers feeding on these plants showed increased mortality compared to those feeding on control plants. Treatment of the plants with sodium selenate did not affect the enzymes involved in the biosynthesis of the plant stress hormones jasmonic acid and salicylic acid, suggesting that the observed insect mortality cannot be attributed to the activation of these hormonal plant defenses. Feeding assays using an artificial diet supplemented with sodium selenate revealed direct toxicity toward N. lugens. With a low concentration of 6.5 +/- 1.5 mu M sodium selenate, half of the insects were killed after 3 days. In summary, sodium selenate treatment of plants can be used as a potential alternative pest management strategy to protect rice against N. lugens infestation through direct toxicity

Evolutionarily conserved and species-specific glycoproteins in the N-glycoproteomes of diverse insect species

N-glycosylation is one of the most abundant and conserved protein modifications in eukaryotes. The attachment of N-glycans to proteins can modulate their properties and influences numerous important biological processes, such as protein folding and cellular attachment. Recently, it has been shown that protein N-glycosylation plays a vital role in insect development and survival, which makes the glycans an interesting target for pest control. Despite the importance of protein N-glycosylation in insects, knowledge about insect N-glycoproteomes is scarce. To fill this gap, the N-glycosites were identified in proteins from three major pest insects, spanning different insect orders and diverging in post-embryonic development, feeding mechanism and evolutionary ancestry: Drosophila melanogaster (Diptera), Tribolium castaneum (Coleoptera) and Acyrthosiphon pisum (Hemiptera). The N-glyco-FASP method for isolation of N-glycopeptides was optimized to study the insect N-glycosites and allowed the identification of 889 N-glycosylation sites in T. castaneum, 941 in D. melanogaster and 1338 in A. pisum. Although a large set of the corresponding glycoproteins is shared among the three insects, species- and order-specific glycoproteins were also identified. The functionality of the insect glycoproteins together with the conservation of the N-glycosites throughout evolution is discussed. This information can help in the elaboration of novel pest insect control strategies based on interference in insect glycosylation

The N-glycome of the hemipteran pest insect Nilaparvata lugens reveals unexpected sex differences

The brown planthopper, Nilaparvata lugens, is a model species for hemimetabolous development and the most important pest insect in rice, which is the major staple crop for about half of the world population. Despite its importance, little is known of the N-glycosylation process in this insect. Here we report on the N-glycome for the post-embryonic stages of N. lugens, revealing unique features that are different from the holometabolous insect models, as the fruit fly Drosophila melanogaster and the beetle Tribolium castaneum. Analysis of the N-glycan fingerprint for male and female adults showed sex-specific N-glycosylation in insects. Specifically, the female adults progress towards a unique glycan profile with a striking increase in high mannose N-glycans. The N-glycome of N. lugens contributes to study pathways differentiating between sexes, and the results shed light on the evolution and differences in development between primitive hemimetabolous insects and more advanced holometabolous insects. The data are discussed in relation to potential function(s) in development and sex specificity

Involvement of OsRIP1, a ribosome-inactivating protein from rice, in plant defense against Nilaparvata lugens

Rice is the most important staple food in the world, but rice production is challenged by several biotic stress factors like viruses, bacteria, fungi and pest insects. One of the most notorious pest insects is Nilaparvata lugens, commonly known as the brown planthopper, which feeds on rice phloem sap and can cause serious damage to rice fields. In order to protect themselves, plants express a wide array of defense proteins such as ribosome-inactivating proteins (RIPs). This study shows that the expression of 'OsRIP1' is highly induced in rice plants infested with N. lugens, with transcript levels more than 100-fold upregulated in infested plants compared to non infested plants. Furthermore, recombinant OsRIP1 was toxic for brown planthoppers when administered through liquid artificial diet. OsRIP1 inactivated insect ribosomes in vitro, suggesting that its toxicity relates to the enzymatic activity of OsRIP1. Over-expression of OsRIP1 in transgenic rice plants did not affect the performance of insects reared on these plants, most likely due to insufficient concentrations of OsRIP1 in the phloem. The data obtained in this research indicate that OsRIP1 can play a role in plant defense against herbivorous insects