Two sympatric Byrsonima species (Malpighiaceae) differ in phenological and reproductive patterns

Sympatric and taxonomically related species may present pre- and/or postzygotic mechanisms for reproductive isolation. We compared the phenology and reproductive biology of Byrsonima intermedia and B. pachyphylla, two sympatric Malpighiaceae species, growing in a remnant of Cerrado in Central Brazil. Both species are evergreens and do not have an overlapping flowering period. In both species, dispersal occurs during the rainy season with low and intermediate overlap in the production of immature and mature fruits, respectively. Both species exhibit self-incompatibility (SI), or, in the case of B. intermedia, facultative SI, and, hence, depend on pollinators to compensate for the lack of spontaneous self-pollination. The flowers of the Byrsonima species were visited by fifteen species of bees that collected pollen and/or oil. Based on their more appropriate intrafloral posture, Epicharis flava and Centris varia were the main pollinators of both species, presenting more frequent visits and/or exhibiting trapline foraging behavior, tending to promote outcrossing. Temporal variation in flowering period and total or partial self-incompatibility seem to function as prezygotic isolation mechanisms that form barriers to gene flow between the studied species and probably avoid competition among pollinators. Both species make oil and pollen available to flower visitors and pollinators almost the entire year, but since Byrsonima intermedia has a long flowering time and is very abundant in the study area, it seems to be more important in maintaining flower-visiting bees.     

Photoperiodism and seasonality in neotropical population of Plutella xylostella L. (Lepidoptera: Yponomeutidae)

Neotropical populations of the diamondback moth Plutella xylostella L. have seasonal cycles of growth and decrease, and moth migration plays a fundamental role in generating such population dynamics. Since the oscillation of these populations is predictable, photoperiod might operate as a signal that triggers the migratory behaviour of the insect. Migration in insects is usually preceded by reproductive diapause, a photoperiodic response that can be characterised by morphological, physiological and behavioural alterations that permit to discriminate between migratory and non-migratory forms. In this study, I tested whether the pre-imaginal and reproductive development of P. xylostella from Minas Gerais (Brazil) is affected by artificial day-lengths that are equivalent to the periods of natural population growth or decrease. No evidence of photoperiodic response was found for the insect reared in laboratory on five different constant photoperiods, from 8h to 16h of light per day. There was no significant variation in survival and duration of egg, larva, and pupa stages or in pupal weight, adult size (forewing length), fecundity, and longevity. Although some species have geographically distinct photoperiodic responses, previous assumptions that cosmopolitan P. xylostella responds to photoperiod in temperate regions was questioned. Migratory and population seasonality among neotropical populations of P. xylostella certainly occurs independently of the photoperiodic announcement of seasonal changes in habitat quality.     

Neuromuscular action of venom from the South American colubrid snake Philodryas patagoniensis

Snakes of the opisthoglyphous genus Philodryas are widespread in South America and cause most bites by colubrids in this region. In this study, we examined the neurotoxic and myotoxic effects of venom from Philodryas patagoniensis in biventer cervicis and phrenic nerve-diaphragm preparations and we compared the biochemical activities of venoms from P. patagoniensis and Philodryas olfersii. Philodryas patagoniensis venom (40 microg/mL) had no effect on mouse phrenic nerve-diaphragm preparations but caused time-dependent neuromuscular blockade of chick biventer cervicis preparations. This blockade was not reversed by washing. The highest concentration of venom tested (40 microg/mL) significantly reduced (p<0.05) the contractures to exogenous acetylcholine (55 microM and 110 microM) and K(+) (13.4 mM) after 120 min; lower concentrations of venom had no consistent or significant effect on these responses. Venom caused a concentration- and time-dependent release of creatine kinase (CK) from biventer cervicis preparations. Histological analysis showed contracted muscle fibers at low venom concentrations and myonecrosis at high concentrations. Philodryas venoms had low esterase and phospholipase A(2) but high proteolytic activities compared to the pitviper Bothrops jararaca. SDS-PAGE showed that the Philodryas venoms had similar electrophoretic profiles, with most proteins having a molecular mass of 25-80 kDa. Both of the Philodryas venoms cross-reacted with bothropic antivenom in ELISA, indicating the presence of proteins immunologically related to Bothrops venoms. RP-HPLC of P. patagoniensis venom yielded four major peaks, each of which contained several proteins, as shown by SDS-PAGE. These results indicate that P. patagoniensis venom has neurotoxic and myotoxic components that may contribute to the effects of envenoming by this species.     

A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field

Establishment of anteroposterior (AP) polarity is one of the earliest decisions in cardiogenesis and plays an important role in the coupling between heart and blood vessels. Recent research implicated retinoic acid (RA) in the communication of AP polarity to the heart. We utilized embryo culture, in situ hybridization, morphometry, fate mapping and treatment with the RA pan-antagonist BMS493 to investigate the relationship between cardiac precursors and RA signalling. We describe two phases of AP signalling by RA, reflected in RALDH2 expression. The first phase (HH4-7) is characterized by increasing proximity between sino-atrial precursors and the lateral mesoderm expressing RALDH2. In this phase, RA signalling is consistent with diffusion of the morphogen from a large field rather than a single hot spot. The second phase (HH7-8) is characterized by progressive encircling of cardiac precursors by a field of RALDH2 originating from a dynamic and evolutionary-conserved caudorostral wave pattern in the lateral mesoderm. At this phase, cardiac AP patterning by RA is consistent with localized action of RA by regulated activation of the Raldh2 gene within an embryonic domain. Systemic treatment with BMS493 altered the cardiac fate map such that ventricular precursors were found in areas normally devoid of them. Topical application of BMS493 inhibited atrial differentiation in left anterior lateral mesoderm. Identification of the caudorostral wave of RALDH2 as the endogenous source of RA establishing cardiac AP fates provides a useful model to approach the mechanisms whereby the vertebrate embryo confers axial information on its organs.     

Immunomagnetic recovery of Mycobacterium bovis from naturally infected environmental samples

AIMS: To adapt an immunomagnetic capture (IMC) technique to concentrate and cultivate Mycobacterium bovis from environmental samples including soil, faeces and urine. METHODS AND RESULTS: Cells of Myco. bovis BCG and wild-type Myco. bovis were successfully isolated and cultured from seeded and naturally infected materials respectively. The IMC cell recovery estimated by colony forming units (CFUs) counts ranged from 0.10% to 0.16% for spiked media, and 0.15-0.36% for naturally infected soil and faeces. Recovery estimated by cell counts calculated using semi-quantitative PCR ranged from 80.3% to 88.6% for spiked and 84.1-88.2% for naturally infected material. The differences in the recovery rates estimated by CFUs compared with pixel intensity is likely to be due to clustering of cells on culture plates, thereby underestimating the true cell count. CONCLUSIONS: The IMC techniques can be applied to isolate viable wild type Myco. bovis from naturally contaminated environmental samples. SIGNIFICANCE AND IMPACT OF STUDY: Cultivation of Myco. bovis from environmental samples using traditional methods is extremely problematic. Here, we demonstrate a novel development of IMC techniques that will greatly facilitate the study of the organism in situ in order to assess its epidemiological importance in bovine tuberculosis persistence.     

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.     

PCR Detection of Oxytetracycline Resistance Genes otr(A) and otr(B) in Tetracycline-Resistant Streptomycete Isolates from Diverse Habitats

A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (Tc^R) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of Tc^R streptomycetes originated from bulk and rhizosphere soil. Fewer Tc^R streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene–carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which Tc^R streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common.