Ultrastructural, Antigenic and Physicochemical Characterization of the Mojuí dos Campos (Bunyavirus) Isolated from Bat in the Brazilian Amazon Region

The Mojuí dos Campos virus (MDCV) was isolated from the blood of an unidentified bat (Chiroptera) captured in Mojuí dos Campos, Santarém, State of Pará, Brazil, in 1975 and considerated to be antigenically different from other 102 arboviruses belonging to several antigenic groups isolated in the Amazon region or another region by complement fixation tests. The objective of this work was to develop a morphologic, an antigenic and physicochemical characterization of this virus. MDCV produces cytopathic effect in Vero cells, 24 h post-infection (p.i), and the degree of cellular destruction increases after a few hours. Negative staining electron microscopy of the supernatant of Vero cell cultures showed the presence of coated viral particles with a diameter of around 98 nm. Ultrathin sections of Vero cells, and brain and liver of newborn mice infected with MDCV showed an assembly of the viral particles into the Golgi vesicles. The synthesis kinetics of the proteins for MDCV were similar to that observed for other bunyaviruses, and viral proteins could be detected as early as 6 h p.i. Our results reinforce the original studies which had classified MDCV in the family Bunyaviridae, genus Bunyavirus as an ungrouped virus, and it may represent the prototype of a new serogroup.

Isoenzymatic variability among five Anopheles species belonging to the Nyssorhynchus and Anopheles subgenera of the Amazon region, Brazil

An isoenzymatic comparative analysis of the variability and genetics differentiation among Anopheles species was done in populations of An. (Nys.) intermedius and An. (Ano.) mattogrossensis of the Anopheles subgenus, and of An. darlingi, An. albitarsis and An. triannulatus of the Nyssorhynchus subgenus, with the aim of detecting differences between both subgenera and of estimating the degree of genetic intere specific divergence. Samples from Macapá, State of Amapá and Janauari Lake, near Manaus, State of Amazonas, were analyzed for eight isoenzymatic loci. Analysis revealed differences in the average number of alleles per locus (1.6-2.3) and heterozygosity (0.060-0.284). However, the proportion of polymorphic loci was the same for An. (Nys.) darlingi, An. (Nys.) triannulatus and An. (Ano.) mattogrossensis (50%), but differed for An. (Nys.) albitarsis (62.5%) and An. (Ano.) intermedius (25%). Only the IDH1 (P > 0.5) locus in all species studied was in Hardy-Weinberg equilibrium. The fixation index demonstrated elevated genetic structuring among species, based on values of Fst = 0.644 and genetic distance (0.344-0.989). Genetic difference was higher between An. (Nys.) triannulatus and An. (Ano.) intermedius (0.989) and smaller between An. (Nys.) albitarsis sensu lato and An. (Nys.) darlingi (0.344). The data show interspecific genetic divergence which differs from the phylogenetic hypothesis based on morphological characters.

Preparation of sand fly (Diptera: Psychodidae: Phlebotominae) specimens for histological studies

Phlebotominae sand fly specimens were prepared for histological and physiological studies. Different fixatives were tested on sectioned and whole bodied adult females in order to obtain good fixation and provide satisfactory penetration of the embedding media. All fixed specimens were infiltrated (up to seven days under 5ºC) and embedded in hydroxyethyl metacrylate. Two-three µm sections were stained, mounted in Canada balsam and observed by light microscopy. Best results were achieved when whole bodied insects were double fixed in Bouin's and Carnoy's fluids (4 h/2 h) and stained in Hematoxilin/Eosin or fixed in calcium formaldehyde and stained in mercury bromophenol blue.

Molecular evidence for a single taxon, Anopheles nuneztovari s.l., from two endemic malaria regions in Colombia

To elucidate the Anopheles nuneztovari s.l. taxonomic status at a microgeographic level in four malaria endemic localities from Antioquia and Córdoba, Colombia, fragments of the cytochrome oxidase subunit I (COI) and the white gene were used. The COI analysis showed low genetic differentiation with fixation index (F ST) levels between -0.02-0.137 and Nm values between 3-∞, indicating the presence of high gene flow among An. nuneztovari s.l. populations from the four localities. The COI network showed a single most common haplotype, type 1 (n = 55), present in all localities, as the likely ancestral haplotype. Analysis of the white gene showed that An. nuneztovari s.l. populations from both departments grouped with haplotypes 19 and 20, which are part of lineage 3 reported previously. The results of the present study suggest that An. nuneztovari s.l. is a single taxon in the area of the present study.

Aedes aegypti on Madeira Island (Portugal): genetic variation of a recently introduced dengue vector

The increasing population of Aedes aegypti mosquitoes on Madeira Island (Portugal) resulted in the first autochthonous dengue outbreak, which occurred in October 2012. Our study establishes the first genetic evaluation based on the mitochondrial DNA (mtDNA) genes [cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4)] and knockdown resistance ( kdr ) mutations exploring the colonisation history and the genetic diversity of this insular vector population. We included mosquito populations from Brazil and Venezuela in the analysis as putative geographic sources. The Ae. aegypti population from Madeira showed extremely low mtDNA genetic variability, with a single haplotype for COI and ND4. We also detected the presence of two important kdr mutations and the quasi-fixation of one of these mutations (F1534C). These results are consistent with a unique recent founder event that occurred on the island of Ae. aegypti mosquitoes that carry kdr mutations associated with insecticide resistance. Finally, we also report the presence of the F1534C kdr mutation in the Brazil and Venezuela populations. To our knowledge, this is the first time this mutation has been found in South American Ae. aegypti mosquitoes. Given the present risk of Ae. aegypti re-invading continental Europe from Madeira and the recent dengue outbreaks on the island, this information is important to plan surveillance and control measures.

Molecular analysis of an odorant-binding protein gene in two sympatric species of Lutzomyia longipalpis s.l.

Lutzomyia longipalpis s.l. is the main vector of American visceral leishmaniasis (AVL) and occurs as a species complex. DNA samples from two Brazilian sympatric species that differ in pheromone and courtship song production were used to analyse molecular polymorphisms in an odorant-binding protein ( obp29 ) gene. OBPs are proteins related to olfaction and are involved in activities fundamental to survival, such as foraging, mating and choice of oviposition site. In this study, the marker obp29 was found to be highly polymorphic in Lu. longipalpis s.l. , with no fixed differences observed between the two species. A pairwise fixation index test indicated a moderate level of genetic differentiation between the samples analysed.

Genetic structure of populations of Pissodes castaneus (De Geer) (Coleoptera, Curculionidae) using amplified fragment length polymorphism

Genetic structure of populations of Pissodes castaneus (De Geer) (Coleoptera, Curculionidae) using amplified fragment length polymorphism. The objective of this study was to determine the genetic structure of populations of Pissodes castaneus from different areas and on different species of Pinus using the PCR-AFLP technique. Twenty samples were analyzed, representing 19 populations from Brazil and one from Florence, Italy, which is the region of origin of P. castaneus. The four combinations of primers generated a total of 367 fragments of DNA, and 100% of polymorphic loci, indicating high degree of molecular polymorphism. The dendrogram did not reveal trends for grouping the populations in relation to origin. The low genetic similarity (0.11 between the most distant groups) and genetic distances of 0.13 and 0.44 for 10 out of the 20 samples may indicate several founding events or multiple introductions of heterogeneous strains into Brazil. The allelic fixation index (Fst) was 0.3851, considered high, and the number of migrants (Nm) was 0.3991, indicating low gene flow among populations. The highest genetic distances were between the population from Irani, SC and Cambará do Sul, RS and Bituruna, PR, indicating an independent founding event or a particular allelic fixation in the former location. The high genetic diversity among populations points out that the populations are genetically heterogeneous with a diverse gene pool in the surveyed areas, what makes them to respond differently to control measures.

Is the capture success of orchid bees (Hymenoptera, Apoidea) influenced by different baited trap designs? A case study from southern Brazil

Orchid bees are increasingly applied on Neotropical biomonitoring and bioindication studies due to the relative easiness of sampling and identification when compared to other bee groups. A considerable number of orchid bee community studies have been adopting baited traps as a sampling method, especially for replication purposes. However, the trap attributes are variable, and hitherto no evaluation of different designs was carried out. Here, five attributes of baited traps were tested: trap volume, number of entrance holes, presence of landing platform, kind of landing platform, and fixation content. We use Mann-Whitney tests to access differences in richness and abundance capture rates for each trap design. We found that volume, number of entrance holes, and fixation content do not influence orchid bees capture. However, the design without landing platforms had a significantly higher capture rate for richness when compared with sanded landing platforms. On the other hand, analyzing the kind of landing platform, we detected a significantly higher richness and abundance for the trap with landing platforms glued with sand. Despite the fact that the effects of different designs tested here were very punctual, we consider that results from samples taken with different baited trap designs are comparable. Some adjustments on trap design can be done according to the particularities of future studies.

Soybean response to starter nitrogen and Bradyrhizobium inoculation on a Cerrado oxisol under no-tillage and conventional tillage systems

In Brazil, Bradyrhizobium inoculation has successfully replaced the use of N fertilizer on soybean [Glycine max (L) Merr.] crops. However, with the expansion of no-tillage cropping systems in the Cerrados region, the idea that it is necessary to use small N rates at the sowing to overcome problems related with N immobilization has become widespread, mainly when soybean is cultivated after a non-legume crop. In this study we examined soybean response to small rates of N fertilizer under no-tillage (NT) and conventional tillage (CT) systems. Four experiments (a completely randomized block with five replicates) were carried out in a red yellow oxisol, during the periods of 1998/1999 and 1999/ 2000, under NT and CT. The treatments consisted of four urea rates (0, 20, 30 and 40 kg ha-1 N). All treatments were inoculated with Bradyrhizobium japonicum strains SEMIA 5080 and SEMIA 5079, in the proportion 1 kg of peat inoculant (1,5 x 10(9) cells g-1) per 50 kg of seeds. In both experiments, soybean was cultivated after corn and the N fertilizer was band applied at sowing. In all experiments, N rates promoted reductions of up to 50 % in the nodule number at 15 days after the emergence. Regardless of the management system, these reductions disappeared at the flowering stage and there was no effect of N rates on either the number and dry weight of nodules or on soybean yields. Therefore, in the Brazilian Cerrados, when an efficient symbiosis is established, it is not necessary to apply starter N rates on soybean, even when cultivated under notillage systems.

Peanut response to lime and molybdenum application in low pH soils

Liming acid soils is considered to assure the availability of Mo in crops. Additionally, in peanuts (Arachis hypogaea L.) the positive response to liming is associated to a better supply of Ca+2, Mo for the nitrogenase-complex activity, and other non-nitrogen fixing activities of the crop. This study was thus undertaken to assess the effect of lime, Mo, and the lime-Mo interaction on peanut crop, on an acid Ultisol at the Mococa Experimental Station, Instituto Agronômico, São Paulo State, Brazil, from 1987 to 1990. A randomized complete block design with four replications, in a 4 x 4 factorial arrangement, was used in the study. The factors included four lime rates (0, 2, 4, and 6 t ha-1) broadcast and incorporated into the soil, and Mo (0, 100, 200, and 300 g ha-1) as (NH4)2MoO4 applied as seed dressing. Lime was applied once at the beginning of the study while Mo was applied at every planting. Peanut seed cv 'tatu' was used. Significant increase in peanut kernel yield with liming was only evident in the absence of Mo, whereas the peanut response to Mo was observed in two out of the three harvests. A higher yield response (28 % increase) was found when Mo was applied without liming. Soil molybdenum availability, as indicated by plant leaf analysis, increased significantly when lime was applied. Molybdenum fertilization led to higher leaf N content, which in turn increased peanut yield in treatments with smaller lime doses.

Effect of fertilizer-N application and seed coating with rhizobial inoculants on soybean yield in eastern Paraguay

Nitrogen removal in soybean grains at harvest may exceed biological N2 fixation, particularly if grain yields are as high as typically achieved on "Terra Rossa" soils of Eastern Paraguay. Applying N fertilizer or coating seeds with rhizobial inoculants that enhance nodulation may represent a way of balancing the N budget. However, the effects of such treatments appear to be highly site-specific. The objective of this study was to examine the effects of N application (N) and rhizobial inoculation (I) on nodulation, N accumulation and soybean yields in Eastern Paraguay. Field experiments were conducted in two consecutive soybean seasons. Dry conditions in the first year delayed sowing and reduced plant number m-2 and pod number plant-1. Grain yields were generally below 2 t ha-1 but the +N+I treatment increased yields by about 75%. In the second year favorable conditions resulted in yields of around 4 t ha-1 and the treatments had no effect. Nitrogen accumulation was higher in the first year and could therefore not explain the observed yield differences between years and treatment combinations. The positive effect of the +N+I treatment in year one was associated with a more rapid root growth which could have reduced susceptibility to intermittent drought stress. Nodule biomass decreased between flowering and pod setting stages in the +I treatment whereas further increases in nodule biomass in the -I treatment may have led to competition for assimilates between nodules and developing pods. Based on these preliminary results we conclude that N application and seed inoculation can offer short-term benefits in unfavorable years without negative effects on yield in favorable years.

Green manure in coffee systems in the region of Zona da Mata, Minas Gerais: characteristics and kinetics of carbon and nitrogen mineralization

The use of green manure may contribute to reduce soil erosion and increase the soil organic matter content and N availability in coffee plantations in the Zona da Mata, State of Minas Gerais, in Southeastern Brazil. The potential of four legumes (A. pintoi, C. mucunoides, S. aterrimum and S. guianensis) to produce above-ground biomass, accumulate nutrients and mineralize N was studied in two coffee plantations of subsistence farmers under different climate conditions. The biomass production of C. mucunoides was influenced by the shade of the coffee plantation. C. mucunoides tended to mineralize more N than the other legumes due to the low polyphenol content and polyphenol/N ratio. In the first year, the crop establishment of A. pintoi in the area took longer than of the other legumes, resulting in lower biomass production and N2 fixation. In the long term, cellulose was the main factor controlling N mineralization. The biochemical characteristics, nutrient accumulation and biomass production of the legumes were greatly influenced by the altitude and position of the area relative to the sun.

Influence of fungicide seed treatment on soybean nodulation and grain yield

Biological N2 fixation is a major factor contributing to the increased competitiveness of Brazilian soybeans on the international market. However, the contribution of this process may be limited by adverse conditions to symbiotic bacteria, such as fungicide seed treatments. This study aimed to evaluate the effects of the fungicides carbendazim + thiram and carboxin + thiram on soybean nodulation, plant growth and grain yield. Two field experiments were carried out in the Cerrado region of the State of Roraima, in a soil with a low organic matter content and no soybean bradyrhizobia. In 2005, seeds were treated with fungicide carbendazim + thiram and commercial inoculants containing the Bradyrhizobium elkanii strains SEMIA 5019 and SEMIA 587 and B. japonicum strains SEMIA 5079 and SEMIA 5080. In 2006, soybean seeds were treated with the fungicides carbendazim + thiram or carboxin + thiram and inoculated separately with each one of the four strains. The plants were evaluated for number of nodules and dry weight, shoot dry weight and total N accumulated in shoots 35 days after plant emergence, while grain yield and N grain content were determined at harvest. Both fungicides reduced soybean nodulation, especially in the presence of B. elkanii strains. The fungicide carbendazim + thiram reduced nodulation by about 50 % and grain yield by more than 20 % (about 700 kg ha-1), in the treatment inoculated with of strain SEMIA 587.

Biodiversity of rhizobia associated with cowpea cultivars in soils of the lower half of the São Francisco River Valley

The biodiversity of rhizobium in soils of the São Francisco Valley is unknown and can be studied using cowpea as trap plants. The objective of this study was to verify the diversity of diazotrophic bacteria that nodulate cowpea in soils of the lower half of the São Francisco River Valley by morphological and genotypic characterization. Seven soil samples (A1, A2, A3, A4, C1, C2 and MC) were collected to capture bacteria associated to five cowpea cultivars (IPA 206, BRS Pujante, BRS Marataoã, Canapu Roxo, and Sempre Verde), in a 5x7 factorial design with three replications. Thirty days after plant emergence, the nodules were collected and the bacteria isolated and analyzed in relation to their growth characteristics in YMA medium. The 581 isolates were grouped in 49 morphologic groups. Of this total, 62.3 % formed colonies in up to three days, 33.4 % grew from the 6th day on, and 4.3 % began to grow 4 to 5 days after incubation. Regarding the formation of acids and alkalis, 63 % acidified the medium, 12 % made it alkaline and 25 % maintained the medium at neutral pH. The highest diversity was observed in the A3 sample and in isolates associated with the cultivars Canapu Roxo and BRS Pujante. Thirty-eight representative isolates were chosen for the genotypic characterization, clustered in four groups based on the restriction analysis of 16s rDNA. This grouping was strongly correlated with the sampling site; 13 rhizobium isolates had an electrophoretic profile distinct from the standard rhizobium strains used in this study.

In-furrow inoculation of soybean as alternative to fungicide and micronutrient seed treatment

Soybean is a major grain crop in Brazil, and yields can be considerably improved by inoculation with selected Bradyrhizobium strains. However, the incompatibility between inoculation and seed treatments with fungicides and micronutrients represents a major barrier to the achievement of high rates of biological N2 fixation. Inoculation practices that can alleviate the negative effects of agrochemicals must therefore be found and in-furrow inoculation seems to be an attractive alternative. This study reports the results of seven field experiments conducted in three growing seasons in Brazil; three in soils previously cropped with inoculated soybean (> 10(4) cells g-1 of soil of Bradyrhizobium), and four in areas where the crop was sown for the first time (< 10² cells g-1 of soil of Bradyrhizobium). The compatibility with fungicides and micronutrients was compared in seeds inoculated with peat or liquid inoculants, or treated with different doses of liquid inoculant in-furrow. In areas with established Bradyrhizobium populations, seed-applied agrochemicals did generally not affect nodulation, but also did not increase yields, while inoculation always increased N grain accumulation or yield, and N fertilizer decreased both nodulation and yield. Where soybean was sown for the first time, the seed treatment with agrochemicals affected nodulation when applied together with peat or liquid inoculant. In-furrow inoculation alleviated the effects of seed treatment with agrochemicals; the best performance was achieved with high Bradyrhizobium cell concentrations, with up to 2.5 million cells seed-1.