A comparative analysis reveals weak relationships between ecological factors and beta diversity of stream insect metacommunities at two spatial levels

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Jellyfish (Chrysaora lactea, Cnidaria, Semaeostomeae) aggregations in southern Brazil and consequences of stings in humans

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Contributions of demography and dispersal parameters to the spatial spread of a stage-structured insect invasion

Stage-structured models that integrate demography and dispersal can be used to identify points in the life cycle with large effects on rates of population spatial spread, information that is vital in the development of containment strategies for invasive species. Current challenges in the application of these tools include: (1) accounting for large uncertainty in model parameters, which may violate assumptions of ‘‘local’’ perturbation metrics such as sensitivities and elasticities, and (2) forecasting not only asymptotic rates of spatial spread, as is usually done, but also transient spatial dynamics in the early stages of invasion. We developed an invasion model for the Diaprepes root weevil (DRW; Diaprepes abbreviatus [Coleoptera: Curculionidae]), a generalist herbivore that has invaded citrus-growing regions of the United States. We synthesized data on DRW demography and dispersal and generated predictions for asymptotic and transient peak invasion speeds, accounting for parameter uncertainty. We quantified the contributions of each parameter toward invasion speed using a ‘‘global’’ perturbation analysis, and we contrasted parameter contributions during the transient and asymptotic phases. We found that the asymptotic invasion speed was 0.02–0.028 km/week, although the transient peak invasion speed (0.03– 0.045 km/week) was significantly greater. Both asymptotic and transient invasions speeds were most responsive to weevil dispersal distances. However, demographic parameters that had large effects on asymptotic speed (e.g., survival of early-instar larvae) had little effect on transient speed. Comparison of the global analysis with lower-level elasticities indicated that local perturbation analysis would have generated unreliable predictions for the responsiveness of invasion speed to underlying parameters. Observed range expansion in southern Florida (1992–2006) was significantly lower than the invasion speed predicted by the model. Possible causes of this mismatch include overestimation of dispersal distances, demographic rates, and spatiotemporal variation in parameter values. This study demonstrates that, when parameter uncertainty is large, as is often the case, global perturbation analyses are needed to identify which points in the life cycle should be targets of management. Our results also suggest that effective strategies for reducing spread during the asymptotic phase may have little effect during the transient phase. Includes Appendix.

Plastic pellets as oviposition site and means of dispersal for the ocean-skater insect Halobates

Microplastics are omnipresent in the oceans and generally have negative impacts on the biota. However, flotsam may increase the availability of hard substrates, which are considered a limiting resource for some oceanic species, e.g. as oviposition sites for the ocean insect Halobates. This study describes the use of plastic pellets as an oviposition site for Halobates micans and discusses possible effects on its abundance and dispersion. Inspection of egg masses on stranded particles on beaches revealed that a mean of 24% (from 0% to 62%) of the pellets bore eggs (mean of 5 and max. of 48 eggs per pellet). Most eggs (63%) contained embryos, while 37% were empty egg shells. This shows that even small plastic particles are used as oviposition site by H. micans, and that marine litter may have a positive effect over the abundance and dispersion of this species. (C) 2012 Elsevier Ltd. All rights reserved.

Mating system parameters at hierarchical levels of fruits, individuals and populations in the Brazilian insect-pollinated tropical tree, Tabebuia roseo-alba (Bignoniaceae)

For many tree species, mating system analyses have indicated potential variations in the selfing rate and paternity correlation among fruits within individuals, among individuals within populations, among populations, and from one flowering event to another. In this study, we used eight microsatellite markers to investigate mating systems at two hierarchical levels (fruits within individuals and individuals within populations) for the insect pollinated Neotropical tree Tabebuia roseo-alba. We found that T. roseo-alba has a mixed mating system with predominantly outcrossed mating. The outcrossing rates at the population level were similar across two T. roseo-alba populations; however, the rates varied considerably among individuals within populations. The correlated paternity results at different hierarchical levels showed that there is a high probability of shared paternal parentage when comparing seeds within fruits and among fruits within plants and full-sibs occur in much higher proportion within fruits than among fruits. Significant levels of fixation index were found in both populations and biparental inbreeding is believed to be the main cause of the observed inbreeding. The number of pollen donors contributing to mating was low. Furthermore, open-pollinated seeds varied according to relatedness, including half-sibs, full-sibs, self-sibs and self- half-sibs. In both populations, the effective population size within a family (seed-tree and its offspring) was lower than expected for panmictic populations. Thus, seeds for ex situ conservation genetics, progeny tests and reforestation must be collected from a large number of seed-trees to guarantee an adequate effective population in the sample.

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.

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.

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.

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.

Influence of total and specific IgE, serum tryptase, and age on severity of allergic reactions to Hymenoptera stings

The aim of this study was to analyze the influence of total serum IgE and other potential risk factors on severity of systemic allergic Hymenoptera sting reactions.

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.

Increased IL-4 and decreased regulatory cytokine production following relocation of Icelandic horses from a high to low endoparasite environment

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland where Culicoides are absent. However, following importation into continental Europe where Culicoides are present, >or=50% of Icelandic horses (1st generation) develop IBH but and allergens induce Th2 responses, we investigated whether horses domiciled in Iceland are Th2-biased, and whether this is determined by helminth infection. We compared the parasite burden and T cell responses between Icelandic horses living in Iceland or Switzerland. Horses in Iceland have higher faecal egg counts, higher tapeworm-specific IgG(T) levels and higher total serum IgE levels than horses in Switzerland. Nevertheless, horses in Iceland displayed a low proportion of IL-4-producing cells in PBMC cultures after polyclonal or parasite extracts stimulation. No IL-4-producing cells were found in PBMC from horses after stimulation by Culicoides extract. Addition of anti-IL-10 and anti-TGF-beta1 to PBMC cultures of horses in Iceland increased the proportion of IL-4-producing cells after polyclonal or parasite antigens stimulation but not stimulation with Culicoides extract. This paralleled the high levels of IL-10 and TGF-beta1 found in supernatants from PBMC cultures of horses in Iceland. Collectively, horses living in Iceland have a high parasite burden but low IL-4 production. This supports the hypothesis that heavy helminth infections have a suppressive effect on IL-4 production mediated by IL-10 and TGF-beta1.