Transmission of Megatrypanum Trypanosomes to Cervus dama by Tabanidae1

Four fallow deer, Cervus dama, became infected with Trypanosoma (Megatrypanum) sp. by oral application of triturated guts from tabanids collected in an area with deer but without any cattle; four control calves remained negative. Upon challenge with triturated guts from tabanids from an area with pastured cattle, the four calves became infected with Trypanosoma (M.) theileri. The prepatent period in deer was five days or less. Haematopota spp. and Tabanus spp. were identified as vectors of the deer trypanosomes. It is concluded that the trypanosomes of C. dama belong to a Megatrypanum species that is not identical with T. theileri.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome’s efferent arm) make fragments after less heat than controls. If motive force and orientation are specified mainly from the organelle-excluding leading front, then endoplasmic streaming in PMN is a catch-up phenomenon, and microtubules do not provide the vector of locomotion but rather stabilize and orient the “baggage” (nucleus, granuloplasm)—i.e., they prevent fishtailing. Moreover, constraints emanating from the centrosome may now be extended to include, maintenance of the motile machinery as an integral part of the cell.     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

Effects of Antifungal Agents Alone and in Combination Against Candida glabrata Strains Susceptible or Resistant to Fluconazole

The rise of Candida spp. resistant to classic triazole antifungal agents has led to a search for new therapeutic options. Here, we evaluated combinations of antifungals in a checkerboard assay against two groups of Candida glabrata strains: one containing fluconazole-susceptible clinical isolates (FS) and another containing fluconazole-resistant laboratory derivative (FR). The most synergistic combination observed was amphotericin B + flucytosine (synergistic for 61.77 % of FS strains and 76.47 % of FR strains). The most antagonistic combination observed was ketoconazole + flucytosine (FS 61.77 % and FR 55.88 %). Surprisingly, most combinations evidenced indifferent interactions, and the best synergism appeared when amphotericin B and flucytosine were combined against both groups of isolates.     

Glandular trichomes in Connarus suberosus (Connaraceae): distribution, structural organization and probable functions

Connarus suberosus is a typical species of the Brazilian Cerrado biome, and its inflorescences and young vegetative branches are densely covered by dendritic trichomes. The objective of this study was to report the occurrence of a previously undescribed glandular trichome of this species. The localization, origin and structure of these trichomes were investigated under light, transmission and scanning electron microscopy. Collections were made throughout the year, from five adult specimens of Connarus suberosus near Botucatu, S?o Paulo, Brazil, including vegetative and reproductive apices, leaves and fruits in different developmental stages, as well as floral buds and flowers at anthesis. Glandular trichomes (GTs) occurred on vegetative and reproductive organs during their juvenile stages. The GTs consisted of a uniseriate, multicellular peduncle, whose cells contain phenolic compounds, as well as a multicellular glandular portion that accumulates lipids. The glandular cell has thin wall, dense cytoplasm (with many mitochondria, plastids and dictyosomes), and a large nucleus with a visible nucleolus. The starch present in the plastids was hydrolyzed during the synthesis phase, reducing the density of the plastid stroma. Some plastids were fused to vacuoles, and some evidence suggested the conversion of plastids into vacuoles. During the final activity stages of the GTs, a darkening of the protoplasm was observed in some of the glandular cells, as a programmed cell death; afterwards, became caducous. The GTs in C. suberosus had a temporal restriction, being limited to the juvenile phase of the organs. Their presence on the exposed surfaces of developing organs and the chemical nature of the reserve products, suggest that these structures are food bodies. Field observations and detailed studies of plant-environment interactions, as well as chemical analysis of the reserve compounds, are still necessary to confirm the role of these GTs as feeding rewards.     

Activity of Combined Antifungal Agents Against Multidrug-Resistant <Emphasis Type="Italic">Candida glabrata</Emphasis> Strains

In this study, we evaluated the in vitro activity of echinocandins, azoles, and amphotericin B alone and in combination against echinocandin/azole-sensitive and echinocandin/azole-resistant Candida glabrata isolates. Susceptibility tests were performed using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute document M27-A3. The checkerboard method was used to evaluate the fractional inhibitory concentration index of the interactions. Cross-resistance was observed among echinocandins; 15% of the isolates resistant to caspofungin were also resistant to anidulafungin and micafungin. Synergistic activity was observed in 70% of resistant C. glabrata when anidulafungin was combined with voriconazole or posaconazole. Higher (85%) synergism was found in the combination of caspofungin and voriconazole. The combinations of caspofungin with fluconazole, posaconazole and amphotericin B, micafungin with fluconazole, posaconazole and voriconazole, and anidulafungin with amphotericin B showed indifferent activities for the majority of the isolates. Anidulafungin combined with fluconazole showed the same percentage of synergism and indifference (45%). Antagonism was detected in 50% of isolates when micafungin was combined with amphotericin B. Combinations of echinocandins and antifungal azoles have great potential for in vivo assays which are required to evaluate the efficacy of these combinations against multidrug-resistant C. glabrata strains.     

Fat body cells of Amblyomma cajennense partially engorged females (Acari: Ixodidae) and their role on vitellogenesis process

During the process of Arthropoda reproduction, the synthesis and uptake of proteins, carbohydrates, and lipids by oocytes is termed vitellogenesis. These compounds that will make up the yolk may be in ticks endogenously synthesized by the oocytes and/or exogenously produced by the fat body and pedicel cells. This study examined the fat body of Amblyomma cajennense ticks at the cytochemical ultrastructural level to demonstrate the presence of lipids, proteins and carbohydrates in trophocytes. The lipids were detected in higher quantity than proteins and carbohydrates in the fat body cells, suggesting that the role of the fat body in tick is stored lipids and carbohydrates to convert them in energy, or still they could be used with cell structural purpose. The electrophoresis technique applied at A. cajennense fat body demonstrated specifically the molecular mass of proteins: about 98 kDa. By the other hands, the fat body is not the organ responsible for the synthesis of the yolk protein, role probably performed by the pedicel cells.     

Comparison Between Etest and Broth Microdilution Methods for Testing Itraconazole-Resistant <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> Susceptibility to Antifungal Combinations

The checkerboard broth microdilution assay (BMD) is the most frequently used method for the in vitro evaluation of drug combinations. However, its use to evaluate the effect of antifungal drugs on filamentous fungi is sometimes associated with endpoint-reading difficulties, and different degrees of interaction are assigned to the same drug combination. We evaluated combinations of the azoles, itraconazole, posaconazole, and voriconazole, with the echinocandins, anidulafungin, caspofungin, and micafungin, against 15 itraconazole-resistant Aspergillus fumigatus clinical strains via the checkerboard BMD and Etest assay. Readings after 24 and 48 h, considering the two reading endpoints, the minimum inhibitory concentration (MIC) and minimum effective concentration (MEC), were performed for both methods. Our results showed that the correlation coefficients between the BMD and Etest methods were quite diverse to the drug combinations tested. The highest correlation coefficients of the Etest with the BMD assays (MEC and MIC reading) were the Etest-MIC reading at 24 h and the Etest-MEC reading at 48 h. Improvements in experimental conditions may increase the correlation between the two methods and ensure that Etest assay can be safely used in the evaluation of antifungal combinations against Aspergillus species.