Reglucosylation of the Benzoxazinoid DIMBOA with Inversion of Stereochemical Configuration is a Detoxification Strategy in Lepidopteran Herbivores

Benzoxazinoids are chemical defenses against herbivores and are produced by many members of the grass family. These compounds are stored as stable glucosides in plant cells and require the activity of glucosidases to release the corresponding toxic aglucones. In maize leaves, the most abundant benzoxazinoid is (2R)-DIMBOA-Glc, which is converted into the toxic DIMBOA upon herbivory. The ways in which three Spodoptera species metabolize this toxin were investigated. (2S)-DIMBOA-Glc, an epimer of the initial plant compound, was observed in the insect frass, and the associated glucosyltransferase activity was detected in the insect gut tissue. The epimeric glucoside produced by the insect was found to be no longer reactive towards plant glucosidases and thus cannot be converted into a toxin. Stereoselective reglucosylation thus represents a detoxification strategy in Spodoptera species that might help to explain their success as agricultural pests on benzoxazinoid-containing crops.

Insect oral secretions suppress wound-induced responses in Arabidopsis

The induction of plant defences and their subsequent suppression by insects is thought to be an important factor in the evolutionary arms race between plants and herbivores. Although insect oral secretions (OS) contain elicitors that trigger plant immunity, little is known about the suppressors of plant defences. The Arabidopsis thaliana transcriptome was analysed in response to wounding and OS treatment. The expression of several wound-inducible genes was suppressed after the application of OS from two lepidopteran herbivores, Pieris brassicae and Spodoptera littoralis. This inhibition was correlated with enhanced S. littoralis larval growth, pointing to an effective role of insect OS in suppressing plant defences. Two genes, an ERF/AP2 transcription factor and a proteinase inhibitor, were then studied in more detail. OS-induced suppression lasted for at least 48 h, was independent of the jasmonate or salicylate pathways, and was not due to known elicitors. Interestingly, insect OS attenuated leaf water loss, suggesting that insects have evolved mechanisms to interfere with the induction of water-stress-related defences.

Role of phytohormones in insect-specific plant reactions

The capacity to perceive and respond is integral to biological immune systems, but to what extent can plants specifically recognize and respond to insects? Recent findings suggest that plants possess surveillance systems that are able to detect general patterns of cellular damage as well as highly specific herbivore-associated cues. The jasmonate (JA) pathway has emerged as the major signaling cassette that integrates information perceived at the plant–insect interface into broad-spectrum defense responses. Specificity can be achieved via JA-independent processes and spatio-temporal changes of JA-modulating hormones, including ethylene (ET), salicylic acid (SA), abscisic acid (ABA), auxin, cytokinins (CK), brassinosteroids (BR) and gibberellins (GB). The identification of receptors and ligands and an integrative view of hormone-mediated response systems are crucial to understand specificity in plant immunity to herbivores.

Specific herbivore-induced volatiles defend plants and determine insect community composition in the field

In response to insect attack, plants release complex blends of volatile compounds. These volatiles serve as foraging cues for herbivores, predators and parasitoids, leading to plant-mediated interactions within and between trophic levels. Hence, plant volatiles may be important determinants of insect community composition. To test this, we created rice lines that are impaired in the emission of two major signals, S-linalool and (E)-β-caryophyllene. We found that inducible S-linalool attracted predators and parasitoids as well as chewing herbivores, but repelled the rice brown planthopper Nilaparvata lugens, a major pest. The constitutively produced (E)-β-caryophyllene on the other hand attracted both parasitoids and planthoppers, resulting in an increased herbivore load. Thus, silencing either signal resulted in specific insect assemblages in the field, highlighting the importance of plant volatiles in determining insect community structures. Moreover, the results imply that the manipulation of volatile emissions in crops has great potential for the control of pest populations.

3-β-d-Glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc) is an insect detoxification product of maize 1,4-benzoxazin-3-ones

In order to defend themselves against arthropod herbivores, maize plants produce 1,4-benzoxazin-3-ones (BXs), which are stored as weakly active glucosides in the vacuole. Upon tissue disruption, BXs come into contact with β-glucosidases, resulting in the release of active aglycones and their breakdown products. While some aglycones can be reglucosylated by specialist herbivores, little is known about how they detoxify BX breakdown products. Here we report on the structure of an N-glucoside, 3-β-d-glucopyranosyl-6-methoxy-2-benzoxazolinone (MBOA-N-Glc), purified from Spodoptera frugiperda faeces. In vitro assays showed that MBOA-N-Glc is formed enzymatically in the insect gut using the BX breakdown product 6-methoxy-2-benzoxazolinone (MBOA) as precursor. While Spodoptera littoralis and S. frugiperda caterpillars readily glucosylated MBOA, larvae of the European corn borer Ostrinia nubilalis were hardly able to process the molecule. Accordingly, Spodoptera caterpillar growth was unaffected by the presence of MBOA, while O. nubilalis growth was reduced. We conclude that glucosylation of MBOA is an important detoxification mechanism that helps insects tolerate maize BXs.

Synergies and trade-offs between insect and pathogen resistance in maize leaves and roots

Determining links between plant defence strategies is important to understand plant evolution and to optimize crop breeding strategies. Although several examples of synergies and trade-offs between defence traits are known for plants that are under attack by multiple organisms, few studies have attempted to measure correlations of defensive strategies using specific single attackers. Such links are hard to detect in natural populations because they are inherently confounded by the evolutionary history of different ecotypes. We therefore used a range of 20 maize inbred lines with considerable differences in resistance traits to determine if correlations exist between leaf and root resistance against pathogens and insects. Aboveground resistance against insects was positively correlated with the plant's capacity to produce volatiles in response to insect attack. Resistance to herbivores and resistance to a pathogen, on the other hand, were negatively correlated. Our results also give first insights into the intraspecific variability of root volatiles release in maize and its positive correlation with leaf volatile production. We show that the breeding history of the different genotypes (dent versus flint) has influenced several defensive parameters. Taken together, our study demonstrates the importance of genetically determined synergies and trade-offs for plant resistance against insects and pathogens.

Silencing OsHI-LOX makes rice more susceptible to chewing herbivores, but enhances resistance to a phloem feeder

The jasmonic acid (JA) pathway plays a central role in plant defense responses against insects. Some phloem-feeding insects also induce the salicylic acid (SA) pathway, thereby suppressing the plant’s JA response. These phenomena have been well studied in dicotyledonous plants, but little is known about them in monocotyledons. We cloned a chloroplast-localized type 2 13-lipoxygenase gene of rice, OsHI-LOX, whose transcripts were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis and the rice brown planthopper (BPH) Niaparvata lugens, as well as by mechanical wounding and treatment with JA. Antisense expression of OsHI-LOX (as-lox) reduced SSB- or BPH-induced JA and trypsin protease inhibitor (TrypPI) levels, improved the larval performance of SBB as well as that of the rice leaf folder (LF) Cnaphalocrocis medinalis, and increased the damage caused by SSB and LF larvae. In contrast, BPH, a phloem-feeding herbivore, showed a preference for settling and ovipositing on WT plants, on which they consumed more and survived better than on as-lox plants. The enhanced resistance of as-lox plants to BPH infestation correlated with higher levels of BPH-induced H2O2 and SA, as well as with increased hypersensitive response-like cell death. These results imply that OsHI-LOX is involved in herbivore-induced JA biosynthesis, and plays contrasting roles in controlling rice resistance to chewing and phloem-feeding herbivores. The observation that suppression of JA activity results in increased resistance to an insect indicates that revision of the generalized plant defense models in monocotyledons is required, and may help develop novel strategies to protect rice against insect pests.

Roots under attack: contrasting plant responses to below- and aboveground insect herbivory

The distinctive ecology of root herbivores, the complexity and diversity of root–microbe interactions, and the physical nature of the soil matrix mean that plant responses to root herbivory extrapolate poorly from our understanding of responses to aboveground herbivores. For example, root attack induces different changes in phytohormones to those in damaged leaves, including a lower but more potent burst of jasmonates in several plant species. Root secondary metabolite responses also differ markedly, although patterns between roots and shoots are harder to discern. Root defences must therefore be investigated in their own ecophysiological and evolutionary context, specifically one which incorporates root microbial symbionts and antagonists, if we are to better understand the battle between plants and their hidden herbivores.

CD4(+)CD25(+) T cells expressing FoxP3 in Icelandic horses affected with insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-beta1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses.

Skin-infiltrating T cells and cytokine expression in Icelandic horses affected with insect bite hypersensitivity: a possible role for regulatory T cells

Equine insect bite hypersensitivity (IBH) is a seasonally recurrent, pruritic skin disorder caused by an IgE-mediated reaction to salivary proteins of biting flies, predominantly of the genus Culicoides. The aim of this study was to define T cell subsets and cytokine profile in the skin of IBH-affected Icelandic horses with particular focus on the balance between T helper (Th) 1, Th2 and T regulatory (Treg) cells. Distribution and number of CD4+, CD8+ and Forkhead box P3 (FoxP3)+ T cells were characterized by immunohistochemical staining in lesional and non-lesional skin of moderately and severely IBH-affected horses (n=14) and in the skin of healthy control horses (n=10). Using real-time quantitative reverse transcription-polymerase chain reaction, mRNA expression levels of Th2 cytokines (Interleukin (IL)-4, IL-5, IL-13), Th1 cytokines (Interferon-gamma), regulatory cytokines (Transforming Growth Factor beta1, IL-10) and the Treg transcription factor FoxP3 were measured in skin and blood samples. Furthermore, Culicoides nubeculosus specific serum IgE levels were assessed. Lesions of IBH-affected horses contained significantly higher numbers of CD4+ cells than skin of healthy control horses. Furthermore, the total number of T cells (CD4+ and CD8+) was significantly increased in lesional compared to non-lesional skin and there was a tendency (p=0.07) for higher numbers of CD4+ cells in lesional compared to non-lesional skin. While the number of FoxP3+ T cells did not differ significantly between the groups, the ratio of Foxp3 to CD4+ cells was significantly lower in lesions of severely IBH-affected horses than in moderately affected or control horses. Interestingly, differences in FoxP3 expression were more striking at the mRNA level. FoxP3 mRNA levels were significantly reduced in lesional skin, compared both to non-lesional and to healthy skin and were also significantly lower in non-lesional compared to healthy skin. Expression levels of IL-13, but not IL-4 or IL-5, were significantly elevated in lesional and non-lesional skin of IBH-affected horses. IL-10 levels were lower in lesional compared to non-lesional skin (p=0.06) and also lower (p=0.06) in the blood of IBH-affected than of healthy horses. No significant changes were observed regarding blood expression levels of Th1 and Th2 cytokines or FoxP3. Finally, IBH-affected horses had significantly higher Culicoides nubeculosus specific serum IgE levels than control horses. The presented data suggest that an imbalance between Th2 and Treg cells is a characteristic feature in IBH. Treatment strategies for IBH should thus aim at restoring the balance between Th2 and Treg cells.

Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity

Salivary gland proteins of Culicoides spp. have been suggested to be among the main allergens inducing IgE-mediated insect bite hypersensitivity (IBH), an allergic dermatitis of the horse. The aim of our study was to identify, produce and characterize IgE-binding salivary gland proteins of Culicoides nubeculosus relevant for IBH by phage surface display technology. A cDNA library constructed with mRNA derived from C. nubeculosus salivary glands was displayed on the surface of filamentous phage M13 and enriched for clones binding serum IgE of IBH-affected horses. Ten cDNA inserts encoding putative salivary gland allergens were isolated and termed Cul n 2 to Cul n 11. However, nine cDNA sequences coded for truncated proteins as determined by database searches. The cDNA sequences were amplified by PCR, subcloned into high level expression vectors and expressed as hexahistidine-tagged fusion proteins in Escherichia coli. Preliminary ELISA results obtained with these fusions confirmed the specific binding to serum IgE of affected horses. Therefore, the putative complete open reading frames derived from BLAST analyses were isolated by RACE-PCR and subcloned into expression vectors. The full length proteins expressed in Escherichia coli showed molecular masses in the range of 15.5-68.7 kDa in SDS-PAGE in good agreement with the masses calculated from the predicted protein sequences. Western blot analyses of all recombinant allergens with a serum pool of IBH-affected horses showed their ability to specifically bind serum IgE of sensitized horses, and ELISA determinations yielded individual horse recognition patterns with a frequency of sensitization ranging from 13 to 57%, depending on the allergen tested. The in vivo relevance of eight of the recombinant allergens was demonstrated in intradermal skin testing. For the two characterized allergens Cul n 6 and Cul n 11, sensitized horses were not available for intradermal tests. Control horses without clinical signs of IBH did not develop any relevant immediate hypersensitivity reactions to the recombinant allergens. The major contribution of this study was to provide a repertoire of recombinant salivary gland allergens repertoire from C. nubeculosus potentially involved in the pathogenesis of IBH as a starting basis for the development of a component-resolved serologic diagnosis of IBH and, perhaps, for the development of single horse tailored specific immunotherapy depending on their component-resolved sensitization patterns.

Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.

Proteome remodelling during development from blood to insect-form Trypanosoma brucei quantified by SILAC and mass spectrometry

Trypanosoma brucei is the causative agent of human African sleeping sickness and Nagana in cattle. In addition to being an important pathogen T. brucei has developed into a model system in cell biology.

Equine insect bite hypersensitivity: immunoblot analysis of IgE and IgG subclass responses to Culicoides nubeculosus salivary gland extract

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of Culicoides and sometimes Simulium spp. The allergens causing IBH are probably salivary gland proteins from these insects, but they have not yet been identified. The aim of our study was to identify the number and molecular weight of salivary gland extract (SGE) proteins derived from Culicoides nubeculosus which are able to bind IgE antibodies (ab) from the sera of IBH-affected horses. Additionally, we sought to investigate the IgG subclass (IgGa, IgGb and IgGT) reactivity to these proteins. Individual IgE and IgG subclass responses to proteins of C. nubeculosus SGE were evaluated by immunoblot in 42 IBH-affected and 26 healthy horses belonging to different groups (Icelandic horses born in Iceland, Icelandic horses and horses from different breeds born in mainland Europe). Additionally, the specific antibody response was studied before exposure to bites of Culicoides spp. and over a period of 3 years in a cohort of 10 Icelandic horses born in Iceland and imported to Switzerland. Ten IgE-binding protein bands with approximate molecular weights of 75, 66, 52, 48, 47, 32, 22/21, 19, 15, 13/12 kDa were found in the SGE. Five of these bands bound IgE from 50% or more of the horse sera. Thirty-nine of the 42 IBH-affected horses but only 2 of the 26 healthy horses showed IgE-binding to the SGE (p<0.000001). Similarly, more IBH-affected than healthy horses had IgGa ab binding to the Culicoides SGE (19/22 and 9/22, respectively, p<0.01). Sera of IBH-affected horses contained IgE, IgGa and IgGT but not IgGb ab against significantly more protein bands than the sera of the healthy horses. The cohort of 10 Icelandic horses confirmed these results and showed that Culicoides SGE specific IgE correlates with onset of IBH. IBH-affected horses that were born in Iceland had IgGa and IgGT ab (p< or =0.01) as well as IgE ab (p=0.06) against a significantly higher number of SGE proteins than IBH-affected horses born in mainland Europe. The present study shows that Culicoides SGE contains at least 10 potential allergens for IBH and that IBH-affected horses show a large variety of IgE-binding patterns in immunoblots. These findings are important for the future development of a specific immunotherapy with recombinant salivary gland allergens.

Evaluation of an in vitro sulphidoleukotriene release test for diagnosis of insect bite hypersensitivity in horses

REASONS FOR PERFORMING STUDY: Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis caused by bites of Culicoides and Simulium species, and improved means of diagnosis are required. OBJECTIVES: The cellular antigen simulation test (CAST) with C. nubeculosus and S. vittatum extracts was assessed in a population of IBH-affected and healthy horses. Variations in test results over a one year period and possible cross-reactivity between different insect extracts was studied. METHODS: A total of 314 mature horses were studied using the CAST. Influence of severity of clinical signs, gender and age were evaluated, and 32 horses were tested repeatedly over one year. The kappa reliability test was used to assess agreement of the test results with different insect extracts. RESULTS: Horses with IBH had significantly higher sLT release than controls with C. nubeculosus and S. vittatum. The highest diagnostic sensitivity and specificity levels were attained when using adult C. nubeculosus extracts with the CAST (78% and 97%, respectively), suggesting that most horses with IBH are sensitised against Culicoides allergens. A proportion of IBH-affected horses was found to be sensitised to allergens of Simulium spp. in addition to those of C. nubeculosus. The CAST with C. nubeculosus had positive and negative predictive values > or = 80% for a true prevalence of IBH of 12-52%. In the follow-up study, the proportion of IBH-affected horses with a positive test result ranged from 90% in November to 68% in March. Severity of clinical signs or age did not influence test results significantly. However, IBH-affected males achieved significantly more positive test results than IBH-affected females. CONCLUSIONS: The CAST with adult C. nubeculosus has high specificity and good sensitivity for diagnosis of IBH. Horses with IBH are mainly sensitised to Culicoides allergens, and some horses are additionally also sensitised to allergens in Simulium spp. POTENTIAL RELEVANCE: The CAST is likely to be a useful test for diagnosis of IBH, even allowing the identification of IBH-affected but asymptomatic horses. This test may also help in further characterisation of allergens involved in this condition.