Potential of the insect growth regulator, fluazuron, in the control of Rhipicephalus sanguineus nymphs (Latreille, 1806) (Acari: Ixodidae): Determination of the LD95 and LD50

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A preliminary study of insect fauna on pig carcasses located in sugarcane in winter in southeastern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A survey of DNA methylation across social insect species, life stages, and castes reveals abundant and caste-associated methylation in a primitively social wasp

DNA methylation plays an important role in the epigenetic control of developmental and behavioral plasticity, with connections to the generation of striking phenotypic differences between castes (larger, reproductive queens and smaller, non-reproductive workers) in honeybees and ants. Here, we provide the first comparative investigation of caste- and life stage-associated DNA methylation in several species of bees and vespid wasps displaying different levels of social organization. Our results reveal moderate levels of DNA methylation in most bees and wasps, with no clear relationship to the level of sociality. Strikingly, primitively social Polistes dominula paper wasps show unusually high overall DNA methylation and caste-related differences in site-specific methylation. These results suggest DNA methylation may play a role in the regulation of behavioral and physiological differences in primitively social species with more flexible caste differences. © 2013 Springer-Verlag Berlin Heidelberg.

Action of the insect growth regulator fluazuron, the active ingredient of the acaricide Acatak®, in Rhipicephalus sanguineus nymphs (Latreille, 1806) (Acari: Ixodidae)

The present study evaluated the efficacy of fluazuron (active ingredient of the acaricide Acatak®) and its effects on Rhipicephalus sanguineus nymphs fed on rabbits exposed to different doses of this insect growth regulator. Three different doses of fluazuron (20 mg/kg, 40 mg/kg, and 80 mg/kg) were applied on the back of hosts (via pour on), while distilled water was applied to the Control group. On the first day of treatment with fluazuron (24 h), hosts were artificially infested with R. sanguineus nymphs. Once fully engorged, nymphs were removed and placed in identified Petri dishes in Biochemical Oxygen Demand (BOD) incubator for 7 days. After this period, engorged nymphs were processed for ultramorphological analysis. The results revealed alterations in the ultramorphology of many chitinous structures (smaller hypostome and chelicerae, less sclerotized scutum, fewer sensilla, fewer pores, absence of grooves, marginal and cervical strips and festoons in the body, even the anal plaque was damaged) that play essential roles for the survivor of ticks and that can compromise the total or partial development of nymphs and emergence of adults after periodic molting. Our findings confirm the efficacy of fluazuron, a more specific and less aggressive chemical to the environment and human health, and that does not induce resistance, in nymphs of the tick R. sanguineus in artificially infested rabbits treated with this arthropod growth regulator (AGR), indicating that it could be used in the control of this stage of the biological cycle of the tick R. sanguineus. © 2013 Wiley Periodicals, Inc.

Sínteses e estudos dos diferentes peptídeos de fusão dos flavivirus causadores da dengue

Pós-graduação em Biotecnologia - IQ

SEASONAL, HABITAT AND SEX-SPECIFIC PATTERNS OF FOOD UTILIZATION BY RED-WINGED BLACKBIRDS (Agelaius phoeniceous) IN EASTERN ONTARIO AND THEIR ECONOMIC IMPORTANCE

Crop depredation by red-winged blackbirds (Agelaius phoeniceus) causes serious economic losses to agricultural crops each year in both Canada and the United States. The concentration of vulnerable, monocultural crops, particularly corn, during periods when large flocks of blackbirds congregate in roosting areas prior to migration has invariably led to heavy feeding pressure (Stone et al., 1972; Wiens and Dyer, 1975; Tyler et al., 1978). Efforts to reduce damage levels by mechanical and chemical dispersal agents have been largely unsuccessful, at least in terms of a long-term solution to the problem. Recently, the lethal control of blackbird populations using surfactants has been proposed. However, the potential repercussions of the removal of substantial numbers of birds from northern breeding areas are virtually unknown (Robertson et al., 1978). Much of the research dealing with the feeding ecology of red-winged blackbirds has been limited to fall and winter periods when large aggregations of birds are actively involved in crop depredation (Goddad, 1969; Williams, 1976; Dolbeer et al., 1978) or pose a potential health hazard (Monroe and Cronholm, 1976). However, what is not known is the degree to which the removal of deleterious weed seed and insect pests cited in several studies (Bird and Smith, 1964; Mott et al., 1972; Robertson et al., 1978) might be of potential value to agriculture. The issue of whether the benefits derived from redwing foraging compensate for the negative aspects associated with crop depredation and health hazards remains largely unresolved. The present study attempted to evaluate the pest status of this species using diet information derived from food habits analysis conducted during the residency of red- winged blackbirds in a northern breeding area. By determining how the feeding ecology of red-winged blackbirds varies on a seasonal basis, among different breeding habitats and between sexes, we hoped to determine more realistically which segments of the population might be responsible for the greatest benefits or detriments and, thereby, more accurately evaluate the economic impact of the species as a whole. To achieve this aim, the study provides an accurate description of the common insects and weed pests utilized by redwings. By determining the relative proportions of those items known to be detrimental, we hoped to illustrate, at least qualitatively, the degree to which redwing foraging is comprised of both beneficial and harmful components.

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the beta-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

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.

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.

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.

Reduced incidence of insect-bite hypersensitivity in Icelandic horses is associated with a down-regulation of interleukin-4 by interleukin-10 and transforming growth factor-beta1

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of insects of the genus Culicoides. IBH does not occur in Iceland due to the absence of Culicoides. However, Icelandic horses exported to mainland Europe as adults (1st generation) have a >/=50% incidence of developing IBH. In contrast, their progeny (2nd generation) has a <10% incidence of IBH. Here we show that peripheral blood mononuclear cells (PBMC) from Icelandic horses born in mainland Europe and belonging either to the IBH or healthy subgroup produce less interleukin (IL)-4 after polyclonal or allergen-specific stimulation when compared with counterparts from horses born in Iceland. We examined a role of IL-10 and transforming growth factor (TGF)-beta1 in down-regulation of IL-4 in healthy 2nd generation Icelandic horses. Supernatants of PBMC from 2nd generation healthy horses down-regulated the proportion of IL-4-producing cells and IL-4 production in stimulated cultures of PBMC from 1st generation IBH. This inhibition was mimicked by a combination of IL-10 and TGF-beta1 but not by the single cytokines. Cultures of stimulated PBMC of healthy 2nd generation horses produced a low level of IL-4, but IL-4 production was increased by anti-equine IL-10 and anti-human TGF-beta1. This shows for the first time that in horses, IL-10 and TGF-beta1 combined regulate IL-4 production in vitro. It is suggested that in this naturally occurring IgE-mediated allergy, IL-10 and TGF-beta1 have a role in the down-regulation of IL-4-induced allergen-specific Th2 cells, thereby reducing the incidence of IBH.

Cloning of IgE-binding proteins from Simulium vittatum and their potential significance as allergens for equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides and sometimes Simulium spp. The aim of this investigation was to identify Simulium allergens associated with IBH. A phage surface display cDNA library expressing recombinant Simulium vittatum salivary gland proteins was screened using sera of IBH-affected horses sensitized to S. vittatum salivary gland proteins as shown in immunoblot, resulting in the identification of seven cDNAs encoding IgE-binding proteins. The deduced amino acid sequences of these proteins showed sequence similarities to antigen 5 like protein (Sim v 1), to a serine protease inhibitor (Sim v 2), to two alpha-amylases (Sim v 3 and Sim v 4), and to three S. vittatum erythema proteins (SVEPs). The cDNA inserts were subcloned and expressed as [His](6)-tagged protein in Escherichia coli and purified using Ni(2+)-chelate affinity chromatography. Mice were immunised with the seven recombinant proteins and the antibodies tested against the recombinant proteins and salivary gland extract (SGE) of S. vittatum and Culicoides nubeculosus in immunoblot analyses. r-Sim v 1 specific mouse Abs recognized a band of about 32 kDa in immunoblots of both S. vittatum and C. nubeculosus SGE, detectable also by serum IgE of IBH-affected horses. Preincubation of horse serum with r-Sim v 1 completely inhibited IgE binding to the 32 kDa band demonstrating the presence of cross-reactive antigen 5 like proteins in both SGE. Determination of IgE levels against the r-Sim v proteins and crude S. vittatum extract by ELISA in sera from 25 IBH-affected and 20 control horses showed that IBH-affected horses had significantly higher IgE levels than controls against r-Sim v 1, 2, 3, 4 and S. vittatum extract, whereas the r-SVEP showed only marginal IgE binding. Further analyses showed that 60% of IBH-affected horses reacted to r-Sim v 1, suggesting that this could be a major allergen for IBH. Forty to twenty percent of the IBH-affected horses reacted with r-Sim v 2, 3 or 4. Combination of the results obtained with the 4 r-Sim v proteins showed that 92% of the IBH-affected but only 15% of the healthy horses had IgE levels against one or more of the 4 r-Sim v proteins. Seventy percent of the healthy horses had detectable IgE against S. vittatum extract, indicating a low specificity of the detection system used. Optimization of the ELISA system will be required to determine reliable cut-off values for the IBH-related allergens. Their in vivo relevance needs to be carefully assessed.

Nucleolar proteins regulate stage-specific gene expression and ribosomal RNA maturation in Trypanosoma brucei

Different life-cycle stages of Trypanosoma brucei are characterized by stage-specific glycoprotein coats. GPEET procyclin, the major surface protein of early procyclic (insect midgut) forms, is transcribed in the nucleolus by RNA polymerase I as part of a polycistronic precursor that is processed to monocistronic mRNAs. In culture, when differentiation to late procyclic forms is triggered by removal of glycerol, the precursor is still transcribed, but accumulation of GPEET mRNA is prevented by a glycerol-responsive element in the 3' UTR. A genome-wide RNAi screen for persistent expression of GPEET in glycerol-free medium identified a novel protein, NRG1 (Nucleolar Regulator of GPEET 1), as a negative regulator. NRG1 associates with GPEET mRNA and with several nucleolar proteins. These include two PUF proteins, TbPUF7 and TbPUF10, and BOP1, a protein required for rRNA processing in other organisms. RNAi against each of these components prolonged or even increased GPEET expression in the absence of glycerol as well as causing a significant reduction in 5.8S rRNA and its immediate precursor. These results indicate that components of a complex used for rRNA maturation can have an additional role in regulating mRNAs that originate in the nucleolus.