An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.

Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Lutz's spontaneous sedimentation technique and the paleoparasitological analysis of sambaqui (shell mound) sediments

Parasite findings in sambaquis (shell mounds) are scarce. Although the 121 shell mound samples were previously analysed in our laboratory, we only recently obtained the first positive results. In the sambaqui of Guapi, Rio de Janeiro, Brazil, paleoparasitological analysis was performed on sediment samples collected from various archaeological layers, including the superficial layer as a control. Eggs of Acanthocephala, Ascaridoidea and Heterakoidea were found in the archaeological layers. We applied various techniques and concluded that Lutz's spontaneous sedimentation technique is effective for concentrating parasite eggs in sambaqui soil for microscopic analysis.

Temporal Geobiotic Mapping: a conceptual mapping technique toward visualising geobiotic areas in cross-section

Geobiota are defined by taxic assemblages (i.e., biota) and their defining abiotic breaks, which are mapped in cross-section to reveal past and future biotic boundaries. We term this conceptual approach Temporal Geobiotic Mapping (TGM) and offer it as a conceptual approach for biogeography. TGM is based on geological cross-sectioning, which creates maps based on the distribution of biota and known abiotic factors that drive their distribution, such as climate, topography, soil chemistry and underlying geology. However, the availability of abiotic data is limited for many areas. Unlike other approaches, TGM can be used when there is minimal data available. In order to demonstrate TGM, we use the well-known area in the Blue Mountains, New South Wales (NSW), south-eastern Australia and show how surface processes such as weathering and erosion affect the future distribution of a Moist Basalt Forest taxic assemblage. Biotic areas are best represented visually as maps, which can show transgressions and regressions of biota and abiota over time. Using such maps, a biogeographer can directly compare animal and plant distributions with features in the abiotic environment and may identify significant geographical barriers or pathways that explain biotic distributions.

Efficiency of the induced mating technique for Toxorhynchites theobaldi (Diptera, Culicidae)

Toxorhynchites mosquitoes play important ecological roles in aquatic microenvironments, and are frequently investigated as potential biological control agents of mosquito disease vectors. Establishment of Toxorhynchites laboratory colonies can be challenging because for some species, mating and insemination either do not occur or require a prohibitive amount of laboratory space for success. Consequently, artificial insemination techniques have been developed to assist with mass rearing of these species. Herein we describe an adapted protocol for colony establishment of T. theobaldi, a species with broad distribution in the Neotropics. The success of the technique and its implications are discussed.

Digital soil mapping: strategy for data pre-processing

The region of greatest variability on soil maps is along the edge of their polygons, causing disagreement among pedologists about the appropriate description of soil classes at these locations. The objective of this work was to propose a strategy for data pre-processing applied to digital soil mapping (DSM). Soil polygons on a training map were shrunk by 100 and 160 m. This strategy prevented the use of covariates located near the edge of the soil classes for the Decision Tree (DT) models. Three DT models derived from eight predictive covariates, related to relief and organism factors sampled on the original polygons of a soil map and on polygons shrunk by 100 and 160 m were used to predict soil classes. The DT model derived from observations 160 m away from the edge of the polygons on the original map is less complex and has a better predictive performance.

Occurrence and Structure of Arbuscular Mycorrhizal Fungal Communities in Cassava after Cultivation of Cover Crops as Observed by the “PCR-DGGE” Technique

ABSTRACT Cassava (Manihot esculenta Crantz) is a highly mycotrophic crop, and prior soil cover may affect the density of arbuscular mycorrhizal fungi (AMFs), as well as the composition of the AMFs community in the soil. The aim of this study was to evaluate the occurrence and the structure of AMFs communities in cassava grown after different cover crops, and the effect of the cover crop on mineral nutrition and cassava yield under an organic farming system. The occurrence and structure of the AMFs community was evaluated through polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A randomized block experimental design was used with four replications. Six different cover crop management systems before cassava were evaluated: black oats, vetch, oilseed radish, intercropped oats + vetch, intercropped oats + vetch + oilseed radish, plus a control (fallow) treatment mowed every 15 days. Oats as a single crop or oats intercropped with vetch or with oilseed radish increased AMFs inoculum potential in soil with a low number of propagules, thus benefiting mycorrhizal colonization of cassava root. The treatments did not affect the structure of AMFs communities in the soil since the AMFs communities were similar in cassava roots in succession to different cover crops. AMFs colonization was high despite high P availability in the soil. The cassava crop yield was above the regional average, and P levels in the leaves were adequate, regardless of which cover crop treatments were used. One cover crop cycle prior to the cassava crop was not enough to observe a significant response in variables, P in plant tissue, crop yield, and occurrence and structure of AMFs communities in the soil. In the cassava roots in succession, the plant developmental stage affected the groupings of the structure of the AMF community.

Euschistus heros mass rearing technique for the multiplication of Telenomus podisi

The objective of this work was to improve the mass rearing technique of Euschistus heros in laboratory. Nymphs and adults were reared at densities 100, 200, 300 and 400 eggs per Petri dish (9 cm diameter), and at 50, 100, 150 and 200 couples per rearing cages (900 mL), respectively. Survival rate of immature stages and survivorship and reproduction of adults were determinated. Survivorship of nymph to adult was the highest (89%) at density 100 eggs per dish. Adult survivorship was independent of density, and 100 couples per cage were the best to improve quality of the produced progeny. In these conditions, fecundity was 160.8±9.28 eggs per female, and a total of 8,950±456 eggs per cage per month was produced. Two hundred couples per cage showed a negative effect on reproduction, which decreased to 65%. With this technique, a colony of 35 cages with 100 couples per cage yields about 313.3 thousands eggs per month, which is enough to supply the egg parasitoid Telenomus podisi to colonize about 35 ha of soybean field.

Chemical and physical composition of grain-type and food-type soybean for food processing

The objective of this work was to evaluate the chemical and physical characteristics of grains of soybean (Glycine max) cultivars for food processing. The soybean cultivars evaluated were: grain-type - BRS 133 and BRS 258; food-type - BRS 213 (null lipoxygenases), BRS 267 (vegetable-type) and BRS 216 (small grain size). BRS 267 and BRS 216 cultivars showed higher protein content, indicating that they could promote superior nutritional value. BRS 213 cultivar showed the lowest lipoxygenase activity, and BRS 267, the lowest hexanal content. These characteristics can improve soyfood flavor. After cooking, BRS 267 cultivar grains presented a higher content of aglycones (more biologically active form of isoflavones) and oleic acid, which makes it proper for functional foods and with better stability for processing, and also showed high content of fructose, glutamic acid and alanine, compounds related to the soybean mild flavor. Because of its large grain size, BRS 267 is suitable for tofu and edamame, while small-grain-sized BRS 216 is good for natto and for soybean sprouts production. BRS 216 and BRS 213 cultivars presented shorter cooking time, which may be effective for reducing processing costs.

Is tagging with visual implant elastomer a reliable technique for marking earthworms?

Visual implant elastomer (VIE) has recently been employed to investigate different aspects of earthworm ecology. However, a number of fundamental questions relating to the detection and positioning of the tag, its persistence and potential effects on earthworms remain unknown. Seven earthworm species belonging to three ecological groupings, with different pigmentation and burrowing behaviour, were tagged using different coloured VIE. External inspection after two days, one week and 1, 10 and 27 months were followed by preservation, dissection and internal inspection. Tags could be seen in living specimens to 27 months, and dissection revealed that in most cases they were lodged in the coelomic cavity, held in place by septa. However, over longer time periods (more than two years), the chlorogogenous tissue tended to bind to the tags and made external observation increasingly difficult. Migration of the VIE material towards the posterior of the earthworm and potential loss of the tag were only observed on rare occasions, and a recovery rate in excess of 98% was recorded. By introducing a reasonable amount of VIE into segments, just after the clitellum, this technique can become a valuable tool in earthworm ecology and life history studies, particularly in short-medium term laboratory and field experiments.