Mutualism from the inside: coordinated development of plant and insect in an active pollinating fig wasp

Recent studies on the obligate interaction between fig trees and their pollinating agaonid wasps have focused on population aspects and wasp-seed exploitation at the level of the inflorescence. Detailed studies on larval and gall development are required to more fully understand how resources are exploited and adaptations fine-tuned by each partner in nursery pollination mutualisms. We studied the larval development of the active pollinating fig wasp, Pegoscapus sp., and the galling process of individual flowers within the figs of its monoecious host, Ficus citrifolia, in Brazil. The pollinator development is strongly dependent on flower pollination. Figs entered by pollen-free wasps were in general more likely to abort. Retained, unpollinated figs had both higher larval mortality and a lower number of wasps. Pegoscapus sp. larvae are adapted to plant development, with two contrasting larval feeding strategies proceeding alongside gall development. The first two larval stages behave as ovary parasites. Later larval stages feed on hypertrophied endosperm. This indicates that a successful galling process relies on endosperm, and also reveals why pollination would be a prerequisite for the production of high-quality galls for this Pegoscapus species.

Sugarwin: A Sugarcane Insect-Induced Gene with Antipathogenic Activity

In sugarcane fields, colonization of the stalk by opportunistic fungi usually occurs after the caterpillar Diatraea saccharalis attacks the sugarcane plant. Plants respond to insect attack by inducing and accumulating a large set of defense proteins. Two homologues of a barley wound-inducible protein (BARWIN), sugarcane wound-inducible proteins SUGARWIN1 and SUGARWIN2, have been identified in sugarcane by an in silico analysis. Antifungal properties have been described for a number of BARWIN homologues. We report that a SUGARWIN:green fluorescent protein fusion protein is located in the endoplasmic reticulum and in the extracellular space of sugarcane plants. The induction of sugarwin transcripts occurs in response to mechanical wounding, D. saccharalis damage, and methyl jasmonate treatment. The accumulation of transcripts is late induced and is restricted to the site of the wound. Although the transcripts of sugarwin genes were strongly increased following insect attack, the protein itself did not show any effect on insect development; rather, it altered fungal morphology, leading to the apoptosis of the germlings. These results suggest that, in the course of evolution, sugarwin-encoding genes were recruited by sugarcane due to their antipathogenic activity. We rationalize that sugarcane is able to induce sugarwin gene expression in response to D. saccharalis feeding as a concerted plant response to the anticipated invasion by the fungi that typically penetrate the plant stalk after insect damage.

Comparing assemblages of Asteraceae and their insect herbivores under different land-use regimens

Accelerated tropical landscape changes occurring over recent decades have produced environmental mosaics comprising remaining isolated green areas and mixed land-use types. Our objective was to study the effects of alterations in the natural landscape on the species composition and structure of assemblages of Asteraceae and their endophagous insects through comparisons between cerrado (savanna), pastures and Eucalyptus stands. We first investigated whether similarities between assemblages of Asteraceae and their insects varied among land uses or localities. Secondly, we asked whether assemblages of Eucalyptus stands and pastures are subsets of those within the cerrado. We sampled within randomly deployed transects in 15 areas. Land use was found to be an important factor in determining plant composition similarity; however, locality did not exert any significant influence. Pastures were less similar to one another, suggesting high beta diversity. Similarities among insect assemblages were correlated with plant assemblage composition, but not with land use or locality. Species of Tephritidae were distributed along localities independently of land use. High beta diversity in Asteraceae assemblages among cerrados and pastures was supported by nestedness analysis. Plant assemblages in Eucalyptus stands were subsets of cerrado, but pasture assemblages were only partial subsets. A higher degree of nestedness in insect assemblages than in plant assemblages indicated lower beta diversity within these herbivores. Our data indicate that many herbivores are specialized on widely distributed plant genera. Conservation of Asteraceae species and their flower head insects depends not only on maintenance of landscape fragments but also on the correct matching of management form and land use. Such management may contribute to reducing isolation of plant and insect species by increasing the connectivity of remaining cerrado tracts, allowing population maintenance even at low abundances.

Analysis of Insect Cuticular Compounds by Non-lethal Solid Phase Micro Extraction with Styrene-Divinylbenzene Copolymers

Insect cuticular hydrocarbons including relatively non-volatile chemicals play important roles in cuticle protection and chemical communication. The conventional procedures for extracting cuticular compounds from insects require toxic solvents, or non-destructive techniques that do not allow storage of subsequent samples, such as the use of SPME fibers. In this study, we describe and tested a non-lethal process for extracting cuticular hydrocarbons with styrene-divinylbenzene copolymers, and illustrate the method with two species of bees and one species of beetle. The results demonstrate that these compounds can be efficiently trapped by ChromosorbA (R) (SUPELCO) and that this method can be used as an alternative to existing methods.

Head-to-Head Comparison of Three Vaccination Strategies Based on DNA and Raw Insect-Derived Recombinant Proteins against Leishmania

Parasitic diseases plague billions of people among the poorest, killing millions annually, and causing additional millions of disability-adjusted life years lost. Leishmaniases affect more than 12 million people, with over 350 million people at risk. There is an urgent need for efficacious and cheap vaccines and treatments against visceral leishmaniasis (VL), its most severe form. Several vaccination strategies have been proposed but to date no head-to-head comparison was undertaken to assess which is the best in a clinical model of the disease. We simultaneously assayed three vaccination strategies against VL in the hamster model, using KMPII, TRYP, LACK, and PAPLE22 vaccine candidate antigens. Four groups of hamsters were immunized using the following approaches: 1) raw extracts of baculovirus-infected Trichoplusia ni larvae expressing individually one of the four recombinant proteins (PROT); 2) naked pVAX1 plasmids carrying the four genes individually (DNA); 3) a heterologous prime-boost (HPB) strategy involving DNA followed by PROT (DNA-PROT); and 4) a Control including empty pVAX1 plasmid followed by raw extract of wild-type baculovirus-infected T. ni larvae. Hamsters were challenged with L. infantum promastigotes and maintained for 20 weeks. While PROT vaccine was not protective, DNA vaccination achieved protection in spleen. Only DNA-PROT vaccination induced significant NO production by macrophages, accompanied by a significant parasitological protection in spleen and blood. Thus, the DNA-PROT strategy elicits strong immune responses and high parasitological protection in the clinical model of VL, better than its corresponding naked DNA or protein versions. Furthermore, we show that naked DNA coupled with raw recombinant proteins produced in insect larvae biofactories -the cheapest way of producing DNA-PROT vaccines-is a practical and cost-effective way for potential "off the shelf" supplying vaccines at very low prices for the protection against leishmaniases, and possibly against other parasitic diseases affecting the poorest of the poor.

Genes involved in thoracic exoskeleton formation during the pupal-to-adult molt in a social insect model, Apis mellifera

Background: The insect exoskeleton provides shape, waterproofing, and locomotion via attached somatic muscles. The exoskeleton is renewed during molting, a process regulated by ecdysteroid hormones. The holometabolous pupa transforms into an adult during the imaginal molt, when the epidermis synthe3sizes the definitive exoskeleton that then differentiates progressively. An important issue in insect development concerns how the exoskeletal regions are constructed to provide their morphological, physiological and mechanical functions. We used whole-genome oligonucleotide microarrays to screen for genes involved in exoskeletal formation in the honeybee thoracic dorsum. Our analysis included three sampling times during the pupal-to-adult molt, i.e., before, during and after the ecdysteroid-induced apolysis that triggers synthesis of the adult exoskeleton. Results: Gene ontology annotation based on orthologous relationships with Drosophila melanogaster genes placed the honeybee differentially expressed genes (DEGs) into distinct categories of Biological Process and Molecular Function, depending on developmental time, revealing the functional elements required for adult exoskeleton formation. Of the 1,253 unique DEGs, 547 were upregulated in the thoracic dorsum after apolysis, suggesting induction by the ecdysteroid pulse. The upregulated gene set included 20 of the 47 cuticular protein (CP) genes that were previously identified in the honeybee genome, and three novel putative CP genes that do not belong to a known CP family. In situ hybridization showed that two of the novel genes were abundantly expressed in the epidermis during adult exoskeleton formation, strongly implicating them as genuine CP genes. Conserved sequence motifs identified the CP genes as members of the CPR, Tweedle, Apidermin, CPF, CPLCP1 and Analogous-to-Peritrophins families. Furthermore, 28 of the 36 muscle-related DEGs were upregulated during the de novo formation of striated fibers attached to the exoskeleton. A search for cis-regulatory motifs in the 5′-untranslated region of the DEGs revealed potential binding sites for known transcription factors. Construction of a regulatory network showed that various upregulated CP- and muscle-related genes (15 and 21 genes, respectively) share common elements, suggesting co-regulation during thoracic exoskeleton formation. Conclusions: These findings help reveal molecular aspects of rigid thoracic exoskeleton formation during the ecdysteroid-coordinated pupal-to-adult molt in the honeybee.

Molecular and biological analysis on the horizontal transfer of insect transposons in Baculoviruses

Summary During the infection of Lepidoptera larvae with baculoviruses the horizontal escape of Tc1-like transposons, termed TCl4.7 and TCp3.2, from the genome of the host Cryptophlebia leucotreta and Cydia pomonella into the genome of Cydia pomonella granulovirus was observed. In this study we addressed the question whether the transposon harboring viruses had a replication advantage over the wild-type and became dominant in the virus population or whether the activity of the host transposable elements is stimulated by virus infection. Biological characterization studies demonstrated that the transposon containing viruses killed C. pomonella larvae slower than CpGV-M. In co-infection experiments of C. pomonella larvae using a mixture of CpGV-M and mutant viruses as inoculum, it was shown that the transposon carrying mutants had a significant selection disadvantage compared to CpGV-M. Transcription levels of the transposase gene of TCp3.2 were investigated in virus infected and uninfected larvae. These experiments demonstrated that a higher level of transposase transcription was detectable in CpGV-M infected than in mock infected control larvae. This observation gave strong evidence that CpGV-M infection might trigger the activity of transposon TCp3.2 within the genome of Cydia pomonella. Our results suggest that the horizontal transfer of insect host transposons into baculovirus genomes might be induced by virus infection.

Comparative phylogeography of two co-distributed arctic-alpine freshwater insect species in Europe

The distribution pattern of European arctic-alpine disjunct species is of growing interest among biogeographers due to the arising variety of inferred demographic histories. In this thesis I used the co-distributed mayfly Ameletus inopinatus and the stonefly Arcynopteryx compacta as model species to investigate the European Pleistocene and Holocene history of stream-inhabiting arctic-alpine aquatic insects. I used last glacial maximum (LGM) species distribution models (SDM) to derive hypotheses on the glacial survival during the LGM and the recolonization of Fennoscandia: 1) both species potentially survived glacial cycles in periglacial, extra Mediterranean refugia, and 2) postglacial recolonization of Fennoscandia originated from these refugia. I tested these hypotheses using mitochondrial sequence (mtCOI) and species specific microsatellite data. Additionally, I used future SDM to predict the impact of climate change induced range shifts and habitat loss on the overall genetic diversity of the endangered mayfly A. inopinatus.rnI observed old lineages, deep splits, and almost complete lineage sorting of mtCOI sequences between mountain ranges. These results support the hypothesis that both species persisted in multiple periglacial extra-Mediterranean refugia in Central Europe during the LGM. However, the recolonization of Fennoscandia was very different between the two study species. For the mayfly A. inopinatus I found strong differentiation between the Fennoscandian and all other populations in sequence and microsatellite data, indicating that Fennoscandia was recolonized from an extra European refugium. High mtCOI genetic structure within Fennoscandia supports a recolonization of multiple lineages from independent refugia. However, this structure was not apparent in the microsatellite data, consistent with secondary contact without sexual incompability. In contrast, the stonefly A. compacta exhibited low genetic structure and shared mtCOI haplotypes among Fennoscandia and the Black Forest, suggesting a shared Pleistocene refugium in the periglacial tundrabelt. Again, there is incongruence with the microsatellite data, which could be explained with ancestral polymorphism or female-biased dispersal. Future SDM projects major regional habitat loss for the mayfly A. inopinatus, particularly in Central European mountain ranges. By relating these range shifts to my population genetic results, I identified conservation units primarily in Eastern Europe, that if preserved would maintain high levels of the present-day genetic diversity of A. inopinatus and continue to provide long-term suitable habitat under future climate warming scenarios.rnIn this thesis I show that despite similar present day distributions the underlying demographic histories of the study species are vastly different, which might be due to differing dispersal capabilities and niche plasticity. I present genetic, climatic, and ecological data that can be used to prioritize conservation efforts for cold-adapted freshwater insects in light of future climate change. Overall, this thesis provides a next step in filling the knowledge gap regarding molecular studies of the arctic-alpine invertebrate fauna. However, there is continued need to explore the phenomenon of arctic-alpine disjunctions to help understand the processes of range expansion, regression, and lineage diversification in Europe’s high latitude and high altitude biota.

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.