Species-specific mitochondrial DNA markers for identification of non-invasive samples from sympatric carnivores in the Iberian Peninsula

Genetic species identification of non-invasively collected samples has become an important tool in ecological research, management and conservation and wildlife forensics. This is especially true for carnivores, due to their elusive nature, and is crucial when several ecologically and phylogenetically close species, with similar faeces, hairs, bones and/or pelts, occur in sympatry. This is the case of the Iberian Peninsula, a region with a carnivore community of 16 species—about two-thirds of the European carnivore fauna. Here we present a simple, efficient and reliable PCR-based protocol, using a novel set of species-specific primers, for the unambiguous identification to species of non-invasively collected samples or forensic materials from Iberian carnivores. For each species, from the consensus of all cytochrome b haplotypes, found here and previously reported, we designed species-specific primer pairs for short fragments, the most likely to persist in low-quantity and degraded DNA samples. The predicted specificity of each primer pair was assessed through PCR of positive DNA extracts from the carnivore species, from an exhaustive array of potential prey and from humans. The robustness of PCR amplification for non-invasively sampled DNA was tested with scat samples. The primers did not produce false positives and correctly identified all carnivore samples to the species level. In comparison with sequencing and PCR-RFLP assays, our method is, respectively, cost- and time-effective, and is especially suited for monitoring surveys targeting multiple populations/species. It also introduces an approach that works for a whole community of carnivores living sympatrically over a large geographic area.     

Antimutagenicity of rosmarinic acid in Swiss mice evaluated by the micronucleus assay

Rosmarinic acid (RA) is a natural phenolic compound which presents different biological activities such as antitumor, antibacterial, anti-inflammatory, hepatoprotective and cardioprotective properties. In view of its important biological activities, the study of the effects of RA on genetic material becomes relevant. Thus, the objective of the present study was to evaluate the mutagenic and/or antimutagenic potential of RA on peripheral blood cells of Swiss mice using the micronucleus assay. Three doses of RA (50, 100 and 200 mg/kg body weight, b.w.) were used for the evaluation of its mutagenic potential. In the antimutagenicity assays, the different concentrations of RA were combined with the chemotherapeutic agent doxorubicin (DXR, 15 mg/kg b.w.). Peripheral blood samples were collected 24, 48 and 72 h after treatment for the evaluation of micronucleated polychromatic erythrocytes (MNPCEs). The results of the mutagenicity assays showed no increase in the frequency of micronuclei in animals treated with different concentrations of RA when compared to the negative controls. Treatment with different concentrations of RA combined with DXR revealed a significant reduction in the frequency of micronuclei compared to animals treated with DXR only. Although the mechanisms underlying the protective effect of RA are not completely understood, the putative antioxidant activity of RA might explain its effect on DXR mutagenicity.     

Seasonal variations in the biochemical composition of the crayfish Parastacus defossus (Crustacea, Decapoda) in its natural environment

The crayfish Parastacus defossus occurs in Uruguay and the state of Rio Grande do Sul in Brazil. It lives in swamps and lakes, where it digs sloped subterranean tunnels that are used as burrows. Because there is little information about the biology, physiology and ecology of this species, the aim of this study was to identify the seasonal variations of its carbohydrate and lipid metabolism. Crayfish were collected monthly (from November 2002 to February 2004) in the Lami region, Porto Alegre municipality (30 degrees 11'41'S-51 degrees 06'00'W). Haemolymph samples, used for determination of glucose, total proteins, triglycerides, total cholesterol and total lipids, were collected in the field using potassium oxalate as an anti-clotting agent. The animals and haemolymph samples were immediately frozen in the field. In the laboratory, the hepatopancreas, gills and abdominal muscles were removed for determination of glycogen, triglycerides, total cholesterol and total lipids. The findings suggest that in P. defossus, lipids are an important reserve of energy used during reproduction in both males and females; whereas glycogen may be used during periods of intense activity or environmental stress.     

Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30–36-month-old, 500–550 kg) with good seminal quality (>80% motile and morphologically normal sperm) had scrotal insulation applied for 5 d. Semen was collected by electroejaculation and cryopreserved at the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1–5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased (P < 0.05) 14 and 21 d after insulation was removed. On 14 and 21 d, the incidence of head defects (9.7 ± 0.6 and 17.0 ± 0.8, respectively; mean ± S.E.M.) was higher (P < 0.05) in agreement with the incidence of nuclear vacuoles (14.0 ± 5.0 and 12.3 ± 2.3) and abnormal chromatin (24.4 ± 7.2 and 30.8 ± 2.8). Although the frequency of cleaved oocytes decreased only on 21 d (P < 0.05), blastocyst rates were lower (P < 0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage (R² = 0.38, 0.48, and 0.30, respectively) and blastocyst rates (R² = 0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates (R² = 0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm.     

Wild Manihot species: botanical aspects, geographic distribution and economic value

A total of 98 Manihot species have been recognized in the genus. All of them are native to the tropics of the New World, particularly Brazil and Mexico. The cultigen, Manihot esculenta Crantz (cassava), grows throughout the lowland tropics. Wild species vary in growth habit from acaulescent or short shrubs to tree-like. Because of their adaptations to different conditions, they are gene reservoirs for tackling many abiotic and biotic stresses such as improving root quality and resistance to diseases. They have been used successfully by the first author for improving protein content, seed-fertility, apomixis, resistance to mealy bug, and tolerance to drought. A table of the most important species from an economic viewpoint is presented.     

Virulence in murine model shows the existence of two distinct populations of Brazilian Vaccinia virus strains

Brazilian Vaccinia virus had been isolated from sentinel mice, rodents and recently from humans, cows and calves during outbreaks on dairy farms in several rural areas in Brazil, leading to high economic and social impact. Some phylogenetic studies have demonstrated the existence of two different populations of Brazilian Vaccinia virus strains circulating in nature, but little is known about their biological characteristics. Therefore, our goal was to study the virulence pattern of seven Brazilian Vaccinia virus strains. Infected BALB/c mice were monitored for morbidity, mortality and viral replication in organs as trachea, lungs, heart, kidneys, liver, brain and spleen. Based on the virulence potential, the Brazilian Vaccinia virus strains were grouped into two groups. One group contained GP1V, VBH, SAV and BAV which caused disease and death in infected mice and the second one included ARAV, GP2V and PSTV which did not cause any clinical signals or death in infected BALB/c mice. The subdivision of Brazilian Vaccinia virus strains into two groups is in agreement with previous genetic studies. Those data reinforce the existence of different populations circulating in Brazil regarding the genetic and virulence characteristics.     

Whole-cell bioconversion of β-sitosterol in aqueous–organic two-phase systems

The selective cleavage of the β-sitosterol side-chain by free Mycobacterium sp. NRRL B-3805 cells was used as a model system for the study of solvent effects in a whole-cell bioconversion in two phase aqueous–organic media. This multi-step degradation pathway leads to the production of 4-androstene-4,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) as a minor product. In an attempt to correlate the substrate and cell partition effects and solvent hydrophobicity (log P) with biocatalytic activity, 15 carboxylic acid esters with log P values between 3 and 10 were screened. The results indicated that the toxicity of the tested solvents in this system could not be correlated to their log P, but seemed to depend on their ability to accumulate in the cells, as these showed a strong affinity towards the organic phase. Different solvent/aqueous ratios and hydrodynamic conditions were further tested in the solvent systems (phthalates) showing significant biodegradation activity. The bioconversion rate was generally not much affected by the stirring speed in the employed range (150–300 rpm) but was strongly influenced by the aqueous/organic phase ratio. Results suggest that the bioconversion takes place at the interphase, its rate being possibly limited by mass transport inside the organic phase.     

Defence,growth and nutrient allocation in the tropical shrub Bauhinia brevipes (Leguminosae)

Herbivory in natural communities can be high, reducing the growth and reproduction of individual plants. To diminish the impact of herbivores, plants use a variety of defensive mechanisms. It is now recognized that the plant world is characterized by a proliferation of secondary metabolites, but models of the evolution of defence strategies assume that plants are constrained by finite resources, and that defences are costly. In the present study, the relationships between defence, growth and nutrient concentration in Bauhinia brevipes Vog. (Leguminosae) in south‐eastern Brazil were investigated. Patterns of herbivory and defence were examined for 170 B. brevipes individuals in 1997 and 1998. Leaf age influenced tannin concentration and herbivore attack on B. brevipes. Mature leaves had higher concentrations of tannins than young leaves, and young leaves were approximately 60% more likely to be attacked than mature leaves. Carbon and nitrogen content explained 36% of the variation in the percentage of attacked leaves in B. brevipes (y = 16.29 – 0.31 C + 0.25 N; r² = 0.36; F = 9.56; P < 0.0001). Tannins acted as defensive compounds on B. brevipes leaves against free‐feeding herbivores, but no clear pattern was observed against galling herbivores. The amounts of nitrogen, carbon, potassium, and aluminium explained approximately 69% of the variation in tannin concentration on B. brevipes leaves (y = 0.62 – 0.16 N – 0.36 K – 0.26 Al + 0.74 C; r² = 0.69; F = 21.18; P < 0.0001). We found an inverse relationship between shoot growth rates and tannin concentration in B. brevipes, indicating that the production of tannins may be costly, therefore offering some evidence for the trade‐off hypothesis. Nevertheless, the weak relationship observed between herbivore damage and tannin concentration indicates that other factors are also involved with herbivore pressure.     

Host plant response and phenotypic plasticity of a galling weevil (Collabismus clitellae: Curculionidae)

The response of a host plant to gall‐inducing insects varies both among and within plants, so that different levels of resources are available to the insects. The weevil Collabismus clitellae Boheman induces galls on the shoots of Solanum lycocarpum St Hil. in south‐east Brazil. Galls are found on a range of parts within an individual plant and are more abundant on smaller plants. In the present study, the host plant response as a possible influence on the performance of C. clitellae both between and within plants was tested. Gall abortion increased with plant height. Within plants, gall size was positively related to shoot diameter and number of chambers within the gall. The increase in gall larval density (number of individuals per gall volume unit) resulted in smaller adults and reduced developmental rates, probably because of resource limitation within the gall. The number of eggs laid by females increased with shoot diameter. Females laid more eggs on thicker shoots, where there are fewer chances to form galls with high larval density. However, this relationship was weak and a large variation was found for adult sizes. The availability of high quality sites is limited to smaller plants and thicker shoots located on the basal region of the plant. The phenotypic plasticity of this insect species in adult size and development time allows individuals growing on low quality sites to reach maturity, thus enhancing exploitation of the host plant.