Leaf anatomy variation within and between three "restinga" populations of Erythroxylum ovalifolium Peyr: (Erythroxylaceae) in Southeast Brazil

Erythroxylum ovalifolium is a woody shrub widespread in the "restinga", i.e. the open scrub vegetation of the Brazilian coastal sandy plains. We examined leaf anatomy variation of this species both within populations and between populations of three "restingas" in the state of Rio de Janeiro. Sites were ca.100 km far from each other and differed in regard to rainfall and vegetation structure: a dry, open site; a wet, dense site and an intermediate one. Microhabitats within sites were: (i) exposed to full irradiance, outside vegetation islands; (ii) partially exposed to full irradiance, at the border of vegetation islands; (iii) shaded, inside vegetation islands. Leaf anatomy parameters were measured for five leaves collected in each of five plants per microhabitat, in each population; they were thickness of the leaf blade, of the palisade and spongy parenchyma, and of the adaxial and abaxial epidermis. Leaves from the dry, open site had narrower abaxial epidermis and a smaller contribution of spongy parenchyma to total leaf blade thickeness than the other two sites, which we attributed to water stress. Adaxial epidermis and leaf are thicker in more exposed microhabitats (i and ii, above), irrespective of site. We proposed that between-site anatomical variation in traits related to water stress, and within-site anatomical variation in traits related to light-use are indicative of ecological plasticity and might help explain the high abundance of E. ovalifolium in the studied populations and along the State of Rio de Janeiro coast.

Leaf optical properties of two liana species Canavalia parviflora Benth. and Gouania virgata Reissk in different light conditions

Lianas are plants that depend on support to reach some appreciable height, and they represent an important structural component of tropical forests. Although they predominate in clearings and gaps, some species survive in the understory. Changes in irradiance between these environments can affect leaf morphology and absorption of photosynthetic active radiation (PAR). We had examined the effects of different light regimes on leaf optical properties, chlorophyll content, specific leaf area, and leaf surface morphology in young seedlings of Canavalia parviflora Benth. (Fabaceae) and Gouania virgata Reissk (Rhamnaceae). The seedlings were distributed on workbenches covered by different layers of neutral shade netting, thus creating three levels of light intensity corresponding to about 40%, 10% and 1.5% of solar irradiance. Plants growing in full sun were used as a control. Both species exhibited an increase in reflectance in full sun and alterations in leaf morphology. Reduction in irradiance induced an increase in absorptance (decrease in reflectance and transmittance) in C. parviflora leaves in the green due to higher chlorophyll content. In G. virgata the spectral leaf changes were less observable. However, the efficiency of absorption was more pronounced in G. virgata than in C. parviflora leaves under 40%, 10% and 1.5% photon flux density (PFD). The greater efficiency of absorption in G. virgata was due to a larger specific leaf area (SLA) under these conditions. The adjustments in leaf optical properties can aid these species in overall carbon gain under limited light conditions.

Harvesting of Protium heptaphyllum (Aubl.) March. seeds (Burseraceae) by the leaf-cutting ant Atta sexdens L. promotes seed aggregation and seedling mortality

The role played by leaf-cutting ants as seed dispersers of non-myrmecochorous plants remains poorly understood. Here we document the harvesting of Protium heptaphyllum (Aubl.) March. seeds (Burseraceae) by the leaf-cutting ant Atta sexdens L. and its consequences for (1) seed deposition pattern; (2) seed germination; and (3) seedling mortality. The study was carried out at Dois Irmãos, a 390 ha reserve of Atlantic forest, northeast Brazil. Ant-seed harvesting on the ground was detected in 18.5% of all fruiting trees and ants harvested 41.1% ± 19.7% of the seed crop (mean ± s). In average, ants piled seeds 3.4 ± 2.2 m away from the trunk of parent trees and seed density in these piles reached 128.8 ± 138.8 seeds 0.25 m² during the peak of seed discarding by ants. During a 13 month period, mean seedling mortality varied from 0.54% up to 10.6% in ant-made seed piles vs. 0.05-4.2% in control samples, what resulted in a total seedling mortality of 97.7% vs. 81%. Ants systematically cut seedling epicotyls, accounting for 55% of seedling mortality in seed piles, whereas only 14 seedlings (4.2%) were cut by ants in the control samples. Our results suggest that seed harvesting by A. sexdens (1) affects approximately 20% of fruiting P. heptaphyllum trees and their seed crops; (2) promotes short-distance seed dispersal and high levels of seed aggregation; and (3) reduces seedling survival beneath parents.

Leaf anatomy of two Anemia Sw. species (Schizaeaceae-Pteridophyte) from a rocky outcrop in Niterói, Rio de Janeiro, Brazil

The ferns Anemia tomentosa (Sav.) Sw. var. anthriscifolia (Schrad.) Mickel and Anemia villosa Humb. & Bonpl. ex Willd. are widely associated with vegetation islands on rocky outcrops in Rio de Janeiro. Both species are desiccation tolerant. The leaf anatomy of these species was examined aiming to identify morphological characteristics that would allow the establishment of these species in water-scarce environments. The plants were harvested on "Pedra de Itacoatiara" and prepared according to the usual procedures. The petiole has a uniseriate epidermis with lignified cell walls, conical stegmata, and uniseriate multicelular and glandular trichomes. In A. villosa, the stomata protrude in a respiratory line. Under the epidermis the cells have thick, lignified walls. The parenchyma has phenolic compounds and starch grains. The petiole vascular bundles are surrounded by endodermis with Casparian strips and the xylem is V-shaped (A. villosa) or arc-shaped (A. tomentosa var. anthriscifolia). The leaf blades have a uniseriate epidermis with sinuous anticlinal and convex periclinal walls, conical stegmata and chloroplasts on both surfaces. The leaf margins of A. villosa have lignified cells. The guard cells of the stomata on the abaxial surface are on the same level or are raised above ordinary epidermal cells. Multicelular uniseriate trichomes and glandular hairs were observed. The dorsiventral mesophyll has loosely packed chlorenchyma with arm-shaped and H-shaped cells. The vascular bundles are surrounded by endodermis with Casparian strips and with parenchymatic extensions towards the epidermis. Anatomical results were analyzed considering the interaction of these plants with abiotic factors.

Leaf damage in a mangrove swamp at Sepetiba Bay, Rio de Janeiro, Brazil

Leaf damage to Rhizophora mangle L., Avicennia schaueriana Stapf. & Leechman, and Laguncularia racemosa L. was studied in a two hectare mangrove swamp site in Sepetiba Bay. Seventeen arthropod morphospecies were identified as being responsible for the damage, and their species diversity was highest on A. schaueriana, followed by R. mangle and L. racemosa. Damage in terms of relative area was greatest in L. racemosa. Almost 9% of mangrove canopy leaf area demonstrated some damage. Loss of leaf area to herbivory was 12.1%, 8.3% and 6.2% in L. racemosa, A. schaueriana and R. mangle respectively. L. racemosa and A. schaueriana showed high percentages of leaf-margin damage in terms of the total damaged leaf area (82.2% and 56.3% respectively), while the most important type of damage in R. mangle was necrosis, representing 58.1% of the total damaged leaf area.

Allelopathic interference of Sapindus saponaria root and mature leaf aqueous extracts on diaspore germination and seedling growth of Lactuca sativa and Allium cepa

Sapindus saponaria (soapberry) is a species that presents a great diversity of chemical compounds, such as saponins; however, few studies have examined the allelopathic effect of this species. Therefore, this study provides an evaluation of the allelopathic potential of aqueous extracts of the roots and mature leaves of S. saponaria on the germination of diaspores and seedlings growth of lettuce (Lactuca sativa) and onion (Allium cepa). The aqueous extract was prepared in the proportion of 100 g of dry plant material in 1,000 mL of distilled water (a concentration of 10% w v-1), and diluted with distilled water to 7.5, 5.0 and 2.5% concentrations. The mature leaf extracts caused delay and decrease in the germination process of the lettuce and onion diaspores, with inhibitory effect concentration-dependent, while the root extracts showed no allelopathic effects on the germination process. Both extracts caused abnormalities and inhibited the growth of shoot and root seedlings.

Leaf and calycine colleters in Odontadenia lutea (Apocynaceae - Apocynoideae - Odontadenieae): their structure and histochemistry

The structure and histochemistry of colleters found on the vegetative and floral apices of Odontadenia lutea are described. Colleters occur on vegetative apices starting at the fourth node, with 68 to 80 colleters being found at each node. Each leaf primordium has only one colleter of axillary origin, 3-5 intra-petiolar, and 12-16 inter-petiolar (intra-stipular). There are four types of colleters: standard, bipartite standard, sessile, and bipartite sessile. Colleters on the reproductive apices alternate with the sepals and are sessile, reduced sessile, tripartite laminar sessile, or asymmetrical. All of the colleters have a central nucleus of parenchymatous cells covered by a palisade uniseriate secretory epidermis and a thin cuticle. Secretory idioblasts were observed in the parenchymatous axis. Vascularization was observed only in standard axillary and laminar colleters. Crystals were observed in the parenchyma of the axillary colleter. Histochemical tests demonstrated that there was no rupturing or distension of the cuticle during the secretion process. Mucilage was identified using the PAS reaction as well as by Mayer's reagent and Ruthenium red staining. The calycine colleters had two distinct secretory phases, the first synthesizing mucilage and the second producing phenolic compounds.

Relationships between crown architecture and available irradiance in two cerrado species with different leaf phenologies

Structural differences between cerrado species with different leaf phenologies are linked to crown architecture, leaf production, and biomass allocation to shoots and leaves. The present study characterized crown structures and the patterns of biomass allocation to leaves and shoots in two woody cerrado species with contrasting leaf phenologies and quantified the irradiance reaching their leaves to determine the best period during the day for photosynthetic activity. The shoots and leaves of five individuals of both Annona coriacea (deciduous) and Hymenaea stigonocarpa (evergreen) were collected along a 50 m transect in a cerrado fragment within the urban perimeter of Catalão - GO, to determine their patterns of biomass allocation in their crowns. The evergreen H. stigonocarpa had significantly higher mean values of shoot inclination (SI), petiole length (PL), leaf area (LA), leaf display index (LDI), and individual leaf area per shoot (ILA), while the deciduous species A. coriacea had significantly higher leaf numbers (LN). The more complex crown of H. stigonocarpa had shoots in more erect positions (orthotropic), with intense self-shading within shoots; A. coriacea, on the other hand, had slanting (plagiotropic) shoots in the crown, allowing similar irradiance levels to all leaf surfaces. The production of plagiotropic shoots by the deciduous species (A. coriacea) is a strategy that enables its use of incident sunlight early in the morning and preventing excessive water loss or excessive irradiance. Hymenaea stigonocarpa (an evergreen), by contrast, had orthotropic shoots and uses intense self-shading as a strategy to avoid excessive irradiance, especially at midday. Differences in crown architectures between evergreen and deciduous species of cerrado sensu stricto can therefore be viewed as adaptations to the environmental light regime.

Preparation of mitotic chromosomes of leaf-cutting ants from the genera Atta and Acromyrmex

Some modifications were made to the methodology of Imai et al. (Jpn. J. Genet. 63: 159-185, 1988) for cytogenetic analysis of the leaf-cutting ants Atta sexdens piriventris and Acromyrmex heyeri (Hymenoptera, Formicidae), shortening preparation time and improving chromosomal preparations. The brain ganglia of prepupae were dissected in a 0.0025% hypotonic solution of colchicine, placed on a glass slide on a cold plate (4 ± 1oC) for 20 min. The material was fixed directly on the cold slide (with cold fixative I), macerated with a histological needle and fixed again with fixative I, followed by fixatives II and III, all of them cold. The slide was flame-dried right after the use of fixative III, and it was allowed to air-dry at room temperature for 2 h. The resulting metaphases presented less contracted chromosomes, with separated and well defined sister chromatids at a high frequency, when the material was processed in the manner described and stained with 3% Giemsa in phosphate buffer (pH 6.8) for 15 min.

Identification of reciprocal hybrids in citrus by the broadness of the leaf petiole wing

Broadness of leaf petiole wing (WB) was investigated as a morphological marker for screening hybrids of the very narrow-winged species Citrus limonia and C. sunki with broad-winged species C. aurantium and C. sinensis. Controlled polinizations produced over 500 reciprocal hybrids with potential in the ongoing rootstock breeding program identified by the Pgi-1 and Prxa-1 isozyme loci. Measurement ratios WB/leaf length, WB/leaf broadness and WB/petiole length identified 86 to 91% of the reciprocal hybrids produced. However, visual classification of WB was an equally efficient but much easier and faster method. It can be very useful in breeding programs when large number of plants have to be screened or when isozyme, RFLP or RAPD laboratories are not available.

Lack of antidiabetic effect of a Eugenia jambolana leaf decoction on rat streptozotocin diabetes

Streptozotocin-diabetic rats were treated for 17 days with a decoction of Eugenia jambolana (Myrtaceae) leaves (15%, w/v) as a substitute for water. Body weight, food and fluid intake, urine volume, glycemia, urinary glucose and urea were evaluated every 5 days. The animals were sacrificed by decapitation and blood samples collected for the determination of glycemia, serum cholesterol, HDL-cholesterol, triglycerides and angiotensin-converting enzyme. The weight of adipose and muscle tissues was also determined. There were no statistically significant differences between treated and untreated rats for any of the biochemical or physiological parameters. We conclude that, at least in this experimental model, Eugenia jambolana leaf decoction has no antidiabetic activity.

Cissampelos sympodialis Eichl (Menispermaceae) leaf extract induces interleukin-10-dependent inhibition of Trypanosoma cruzi killing by macrophages

The aqueous fraction of the ethanolic extract (AFL) of Cissampelos sympodialis Eichl (Menispermaceae), popularly known as milona, has been shown to have both immunosuppressive and anti-inflammatory effects. In the present study we investigated the modulation of macrophage antimicrobicidal activity by in vitro treatment with the extract from C. sympodialis. Normal and thioglycolate-elicited mouse peritoneal macrophages were infected in vitro with the protozoan Trypanosoma cruzi DM28c clone. We observed that the AFL (used at doses ranging from 13 to 100 µg/ml) increased T. cruzi growth and induced a 75% reduction in nitric oxide production. This inhibition could be mediated by the stimulation of macrophage interleukin-10 (IL-10) secretion since the in vitro treatment with the AFL stimulated IL-10 production by T. cruzi-infected macrophages. These results suggest that the anti-inflammatory effect of the AFL from C. sympodialis could be, at least in part, mediated by the inhibition of macrophage functions and that the inhibition of macrophage microbicidal activity induced by the C. sympodialis extract may be mediated by the decrease in macrophage function mediated by interleukin-10 production.

Low variation in ribosomal DNA and internal transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae)

Leaf-cutting ants of the genera Atta and Acromyrmex (tribe Attini) are symbiotic with basidiomycete fungi of the genus Leucoagaricus (tribe Leucocoprineae), which they cultivate on vegetable matter inside their nests. We determined the variation of the 28S, 18S, and 5.8S ribosomal DNA (rDNA) gene loci and the rapidly evolving internal transcribed spacers 1 and 2 (ITS1 and ITS2) of 15 sympatric and allopatric fungi associated with colonies of 11 species of leafcutter ants living up to 2,600 km apart in Brazil. We found that the fungal rDNA and ITS sequences from different species of ants were identical (or nearly identical) to each other, whereas 10 GenBank Leucoagaricus species showed higher ITS variation. Our findings suggest that Atta and Acromyrmex leafcutters living in geographic sites that are very distant from each other cultivate a single fungal species made up of closely related lineages of Leucoagaricus gongylophorus. We discuss the strikingly high similarity in the ITS1 and ITS2 regions of the Atta and Acromyrmex symbiotic L. gongylophorus studied by us, in contrast to the lower similarity displayed by their non-symbiotic counterparts. We suggest that the similarity of our L. gongylophorus isolates is an indication of the recent association of the fungus with these ants, and propose that both the intense lateral transmission of fungal material within leafcutter nests and the selection of more adapted fungal strains are involved in the homogenization of the symbiotic fungal stock.

Inotropic effect of Citrus sinensis (L.) Osbeck leaf extracts on the guinea pig atrium

The objective of the present investigation was to determine the contractile effect of crude and acetone leaf extracts of Citrus sinensis (L.) Osb. on mammalian myocardium. Crude leaf extracts have been used in folk medicine to treat neurological disorders. Some flavonoids isolated from this plant presented a positive inotropic effect on myocardium. This motivated us to test the extracts on the atria of guinea pigs of both sexes (300-500 g) and surprisingly we observed inotropic depression instead of an increase in force. The maximum effect of the crude extract was 79.4 ± 8.1% of the control force amplitude (N = 5 hearts, 10 trials, 27 ± 0.1ºC, stimulus: 2 Hz, 400 V, 0.5 ms). The EC50 for crude, ethanol, acetic, aqueous, and acetone extracts was 300, 300, 600, 1000, and 140 µg/ml, respectively, with a Hill constant of 1.8, 2.0, 2.5, 2.0, and 1.4, respectively. Blockade of cholinergic, beta-adrenergic, or opioid membrane receptors with 1.5 µM atropine sulfate, 1 µM propranolol, and 10 µM naloxone, respectively, did not change the effect of the crude extract. The acetone extract abolished the Bowditch positive staircase phenomenon (N = 5 hearts, 10 trials, 27 ± 0.1ºC), suggesting a possible reduction of the calcium inward current, and also promoted the so-called Woodworth phenomenon. The effect was concentration-dependent and indicated the existence of another inhibitory contractile mechanism such as the simultaneous activation of some of the membrane potassium channels reducing the myocardial action potential duration and further decreasing the cellular calcium entry.

Ginkgo biloba leaf extract (EGb 761) enhances catalepsy induced by haloperidol and L-nitroarginine in mice

Ginkgo biloba extract EGb 761 has been reported to have therapeutic effects which have been attributed to anti-oxidant and free radical-scavenging activities, including a direct action on nitric oxide production. L G-nitro-arginine (L-NOARG), a nitric oxide synthase inhibitor, and haloperidol, a drug that blocks dopamine receptors, are both known to induce catalepsy in rodents. Nitric oxide has been shown to influence dopaminergic transmission in the striatum. The purpose of the present study was to evaluate the effect of the extract obtained from leaves of Ginkgo biloba tree EGb 761 on catalepsy induced by haloperidol or by L-NOARG. Albino Swiss mice (35-45 g, N = 8-12) received by gavage a single or repeated oral dose (twice a day for 4 days) of EGb 761 followed by ip injection of haloperidol or L-NOARG. After the treatments, the animals were submitted to behavioral evaluation using the catalepsy test. Acute treatment with 80 mg/kg EGb did not modify the catalepsy induced by L-NOARG but, the dose of 40 mg/kg significantly enhanced haloperidol-induced catalepsy measured at the 10th min of the test. After repeated treatment with 80 mg/kg EGb 761, a significant increase in the cataleptic effect produced by both haloperidol and L-NOARG was observed. These data show that repeated EGb 761 administration increases the effects of drugs that modify motor behavior in mice. Since the catalepsy test has predictive value regarding extrapyramidal effects, the possibility of pharmacological interactions between haloperidol and Ginkgo biloba extracts should be further investigated in clinical studies.