Purificação, caracterização e atividade bioinseticida de um inibidor de tripsina de sementes de Crotalaria pallida

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulphate fractionation, affinity chromatography on immobilized Trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named ITC, had Mr of 32.5 kDa by SDS-PAGE and was composed by two subunits with 27.7 and 5.6 kDa linked by disulphide bridges, a typical characteristic of Kunitz-Inhibitor family. ITC was stable until 50°C, and at 100°C its residual activity was of about 60%. Also, ITC was stable at pHs 2 to 12. The inhibition of trypsin by ITC was non-competitive, with a Ki of 8,8 x 10-7M. ITC inhibits weakly other serine proteinases such as chymotrypsin and elastase. The inhibition of papain (44% of inhibition), a cysteine proteinase was an indicative of the bi-functionality of ITC. In vitro assays against digestive proteinases from several Lepdoptera, Diptera and Coleoptera pests were made. ITC inhibited in 100% digestive enzymes of Ceratitis capitata (fruit fly), Spodoptera frugiperda and Alabama argillacea, the last one being a cotton pest. It also inhibited in 74.4% Callosobruchus maculatus (bean weevil) digestive enzymes, a Coleoptera pest. ITC, when added in artificial diet models, affected weakly the development of C. capitata larvae and it had a WD50 of 2.65% to C. maculatus larvae

High performance liquid chromatography determined alkamide levels in Australian-grown Echinacea spp.

Extracts of Echinacea spp. are widely used as therapeutic immunostimulants with such activity being attributed in part to the alkamide fractions of these plants. Using high performance liquid chromatography, the levels of 8 alkamides, including 2 tetraene alkamides (dodeca-2E, 4E, 8Z, 10E/Z-tetraenoic acid isobutylamide), were quantitatively determined in 2 Australian-grown Echinacea spp. Overall, the levels of alkamides in Australian-grown E. angustifolia were found to be comparable with levels obtained in this study and other studies for USA and European Echinacea spp. However, results obtained for one sample of E. angustifolia suggested that it may have been mislabelled and that it was most likely a sample of E. pallida. Levels of tetraene alkamides in Australian-grown E. purpurea were also similar to, if not higher, than levels which have been reported for the same species grown in Germany and the USA. Preliminary studies on the stability of alkamide compounds in E. angustifolia indicated that they are susceptible to degradation, with a 13% loss of alkamide level over 2 months. Overall, results indicate that there is considerable potential to develop Echinacea as a viable crop in Australia.

Responses of two species of mesquite to initial and follow-up applications of selected herbicides in a potted trial

Quilpie mesquite (Prosopis velutina) is an invasive woody weed that is believed to have been introduced into south-west Queensland in the 1930s. Following the withdrawal of 2,4,5-T, research on P. pallida resulted in revised recommendations for control of all Prosopis spp. in Queensland. Adoption of many of these recommendations for Quilpie mesquite control produced substandard results. Following a pilot trial, a shade-house experiment was conducted to determine the differences in susceptibility of two species of mesquite, P. velutina and P. pallida, to commonly available herbicides. It was hypothesized that P. velutina was less susceptible than P. pallida, based upon claims that the registered chemical recommendations for Prosopis spp. were not sufficiently effective on P. velutina. Nine foliar herbicide treatments were applied to potted shade-house plants. Treatment effects indicated differing susceptibility between the two species. P. velutina consistently showed less response to metsulfuron, fluroxypyr, 2,4-D/picloram and triclopyr/picloram, compared to the glyphosate formulations, where negligible differences occurred between the two species. The response to glyphosate was poor at all rates in this experiment. Re-application of herbicides to surviving plants indicated that susceptibility can decrease when follow-up application is in autumn and the time since initial application is short. The relationship between leaf structure and the volume of spray adhering to a plant was assessed across species. The herbicide captured by similar-sized plants of each species differed, with P. pallida retaining a greater volume of herbicide.

Diversidad florística del bosque de galería en dos localidades del departamento de Carazo, Nicaragua

En las localidades Los Encuentros y Chacocente, en el departamento de Carazo, se realizó un estudio sobre la composición florísticas de las especies arbóreas en el bosque de galería. Los resultados corresponden a un inventario completo de los árboles de 1 O cm de diámetro a la altura del pecho (Dap) y regeneración natural en parcelas con área neta de muestreo de 1 hectárea en cada sitio, con sub-parcelas de 100 m2 para el muestreo de regeneración. Los bosques estudiados son Chacocente (360 msnm) y Los Encuentros (180 msnm). Ambos bosques son de galería y pertenecen a la zona de Vida Bosque Tropical Seco con transición a subtropical. , según el sistema de clasificación de Holdridge. En ambos bosques se encontraron un total 65 especies, pertenecientes a 39 familias y 53 géneros. En el bosque localizado en Los Encuentros, se encontraron 24 especies, siendo las mas frecuentes el Papaturro (Coccoloba caracasana), anona (Annona glabra) y Chaperno blanco (Lonchocarpus minimiflorus). En Chacocente se encontraron 41 especies, siendo las mas frecuentes Palo de piojo (Trichilia hirta), Naranjillo (Capparis odoratisima) y Melero (Thoudinium decandrum). La abundancia de árboles con Dap mayor de 10 cm fue de 333 en Chacocente y 379 en Los Encuentros. Los cocientes de mezcla obtenidos fueron 1:8 para el bosque del Refugio de Vida Silvestre, Chacocente y 1:15 para el de los Encuentros. En ambas comunidades difieren las familias mayormente representadas. Los valores de los coeficientes de Jaccard (4.4 %) y Sorensen (8.4 %) revelan poca similitud florística entre ambos sitios. La regeneración natural presentó en algunos aspectos el mismo comportamiento de la vegetación con Dap mayor o igual a los 10 cm, siendo la única diferencia los valores del coeficiente de mezcla que presentan al bosque de la comunidad Los Encuentros con mayor diversidad florística (l :50 en comparación con 1:16 de Chacocente). La abundancia de individuos de la regeneración natural fue de 242 individuos por hectárea en Los Encuentros y de 860 individuos por hectarea en Chacocente.

Some freshwater ostracods (Crustacea: Ostracoda) from South Wales

As background to a study of the application of astracods in environmental archaeology, a number of sites in South Wales were visited and sampled. Sites included seven broad environmental categories consisting of lakes, permanent ponds, non-permanent ponds, semi-static canals and reens (drainage ditches), non-permanent small lotic water-bodies, permanent fast-flowing waters and wells. In all, twenty-three species were recorded, and with one exception all belonged to the predominately freshwater Cypridoidea. Overall the most commonly encountered species in South Wales was Cypria ophthalmica. Comparing finds with earlier records, it would appear that Ilyocypris bradyi, Candona pratensis, Eucypris lilljeborgi, Herpetocypris chevreuxi, Potamocypris variegata, P. similis and P. pallida are new additions to the Welsh fauna.

Biopotentials of marine sponges from China oceans: past and future

An extensive literature survey of over 17 Journals was carried out on Chinese sponges and their natural products in the period from 1980 to 2001. This review is thus intended to provide the first thorough overview of research on marine sponges from China Ocean territories. Information is provided about the rather-limited taxonomic study of Chinese marine sponges, with an analysis on their distribution and diversity. Research findings on the natural products and their bioactivity screening from Chinese sponges are summarized. The weaknesses, gaps and problems in the past R&D program of Chinese sponges are identified, which point to the future opportunities in exploiting these huge untapped sponge resources. The report is expected to serve as an entry point for understanding Chinese sponges and for furthering R&D on their bioactive compounds for new drug development. (C) 2003 Elsevier Science B.V. All rights reserved.

微型浮游动物及其对浮游植物的摄食压力

本论文研究了渤海，胶州湾和东海20-200 μm微型浮游动物（桡足类幼虫、纤毛虫）的数量和生物量（1% Lugol's 试剂固定1000ml，Utermohl法150倍显微镜检查），在渤海和东海用稀释法（Landry and Hassett, 1982; Burkill et al., 1990）估计了微型浮游动物（<200 μm）对浮游植物的摄食压力。对上述海区调查的时间如下：渤海：1997年6，1998年6月（莱州湾），1998年10月；胶州湾1997年8、11月，1998年2、4、8、11月，1999年2月；东海：1998年7月。结果如下：渤海：1997年6月，只发现一种砂壳纤毛虫，数量为0-980个/L。1998年6月（莱州湾），微型浮游动物数量为30-2390个/L，生物量为1.5-25 μg C/L，水体中生物量为0.1-53.2 mgC m~(-2)。共有13种砂壳纤毛虫和一种无壳纤毛虫。纤毛虫的数量为0-2380个/L，生物量为0-24.52 μg C/L。纤毛虫优势种有无壳纤毛虫、Tintinnopsis pallida、T. amoyensis和T. chinglanensis。微型浮游动物的各个类群呈斑块分布。1998年10月，微型浮游动物的数量为0-770个/L，生物量为0-12.3 μg C/L，水体中生物量为0-136 mgC m~(-2)。在两次大面调查间隔的6天内，发生大风。两次大面站调查海区的温盐分布格局没有很大变化，但微型浮游动物的变化却十分显著，这也反映了恶劣天气对海洋生物的影响。在几个连续站对微型浮游动物的连续监测表明，微型浮游动物的分布与水团有很大关系。胶州湾：桡足类幼虫在8月数量最大（850个/L）。共记录两种无壳纤毛虫和7种砂壳纤毛虫。微型浮游动物数量弯化很大，从10-22600个/L。微型浮游动物的分布格局可能是湾内和湾外海水混合的结果：1997年11月，无壳纤毛虫No.12从湾外涌入，其他航次无壳纤毛虫No.11在湾内发生，向湾外扩散。东海：微型浮游动物数量较少，最大数量为1970个/L，绝大多数站位低于300个/L，生物量为0.08-45 μg C/L，水体50m积分生物量为5-283mgC m~(-2)。微型浮游动物有明显的斑块分布，连续监测没有发现明显的变化。根据稀释培养的结果，微型浮游动物对浮游植物摄食压力如下所述：渤海三个航次的微型浮游动物对浮游植物摄食率为0.13-1.01d~(-1)，每天摄食浮游植物生物量（现存量）的12-49%，初级生产力的34-267%。东海稀释培养的结果为微型浮游动物地浮游植物的摄食率为0.2-1.2d~(-1)，每天摄食浮游动物生物量的20-71%，摄食初级生产力的54-98%。由于稀释培养实验的设计和操作有许多争议之外，本文只将结果列出，对方法不作讨论。

Summertime ciliate and copepod nauplii distributions and micro-zooplankton herbivorous activity in the Laizhou Bay, Bohai Sea, China

The abundance and biomass of ciliated protozoa and copepod nauplii were investigated at 21 grid stations and two anchored stations in the Laizhou Bay, Bohai Sea, China in June 1998. Dilution incubations were carried out to investigate micro-zooplankton grazing pressure at the anchored stations during spring tide and neap tide. The dominant species were Tintinnopsis amoyensis, T. chinglanensis, T. pallida and aloricate ciliates. A total of 13 species of tintinnids were found. The total abundance of ciliates and nauplii ranged from 30 to 2390 ind l(-1) at grid stations. Tintinnopsis amoyensis was the only ciliate found at the anchored stations and in concentrations which varied from 0 to 6700 ind l(-1). The spatial distribution of ciliates was patchy. Tintinnopsis amoyensis and T. pallida were distributed in the Weihe River mouth and Xiaoqinghe River mouth respectively. The aloricate ciliates, T. chinglanensis and Codonellopsis ostenfeldi dominated offshore in sequence. The water mixing process may affect the spatial pattern of the dominant ciliate species. The abundance and biomass of copepod nauplii were in the range of 0-140 ind l(-1) and 0-7 mu g C l(-1) respectively, with the peak appearing at grid station 15. The total biomass of ciliates and copepod nauplii was in the range of 1(.)5-25 mu g C l(-1). Water column biomass of ciliates and nauplii varied from 2(.)37 to 52(.)3 mg C m(-2). At the anchored stations, the phytoplankton growth rates ranged from undetectable to 0 21 d(-1) and micro-zooplankton grazing rates from 0 13 to 0(.)57 d(-1). The grazing pressure of micro-zooplankton were 12 to 43% of the chlorophyll standing stock and 84 to 267% of the chlorophyll (C) 2000 Academic Press.

Modelado de la dinámica y producción forestal de la Selva Pedemontana de Yungas, Argentina

Las Yungas representan el ecosistema de Selvas subtropicales de mayor extensión de la Argentina, constituyendo un área de gran biodiversidad y de recursos madereros. En ella la explotación forestal fue intensa debido a su relieve de pendiente suave, cercanía a centros urbanos y a la creciente demanda de productos madereros causando una gran fragmentación y comprometiendo su resiliencia. La sustentabilidad del manejo forestal se basa en el conocimiento de la composición florística, del crecimiento, la dinámica poblacional y la proyección en el tiempo de la evolución del bosque. Los objetivos planteados en esta tesis fueron determinar la composición y estructura forestal, definir las curvas de crecimientos, determinar las tasas de reclutamiento y mortalidad de las especies de valor, y proyectar la evolución de la Selva en distintas situaciones de manejo. Los parámetros estructurales muestran que las especies dominantes son Anadenanthera colubrina var cebil, Phyllostilon rhamnoides, Trichilia hieronymi, Parapiptadenia excelsa y Calycophyllum multiflorum. Las bajas tasas de incorporaciones y existencias de las especies de mayor valor, Cedrela balansae, Myroxylon peruiferum y Amburana cearensis, determinan su vulnerabilidad. La dinámica está determinada por la formación de claros que generan cambios en las condiciones de luz. Las variables que influyen sobre el crecimiento diamétrico son el tamaño del individuo, la posición sociológica y la densidad del rodal. Las especies de mayores crecimientos diamétricos son C. balansae, A. colubrina var. cebil, P. excelsa, Astronium urundeuva y M. peruiferum siendo el tiempo estimado en lograr sus diámetros mínimos de corta (DMC) de 50, 35, 60, 55 y 80 años respectivamente. La reducción de la densidad del rodal favorece los incrementos diamétricos de los individuos de las especies de valor forestal. La proyección del rodal demuestra que un aprovechamiento forestal realizado con las condiciones técnicas y DMC vigentes, la estructura, la dinámica y el volumen aprovechable no se recupera en un período de 25 años.

Modelado de la dinámica y producción forestal de la Selva Pedemontana de Yungas, Argentina

Las Yungas representan el ecosistema de Selvas subtropicales de mayor extensión de la Argentina, constituyendo un área de gran biodiversidad y de recursos madereros. En ella la explotación forestal fue intensa debido a su relieve de pendiente suave, cercanía a centros urbanos y a la creciente demanda de productos madereros causando una gran fragmentación y comprometiendo su resiliencia. La sustentabilidad del manejo forestal se basa en el conocimiento de la composición florística, del crecimiento, la dinámica poblacional y la proyección en el tiempo de la evolución del bosque. Los objetivos planteados en esta tesis fueron determinar la composición y estructura forestal, definir las curvas de crecimientos, determinar las tasas de reclutamiento y mortalidad de las especies de valor, y proyectar la evolución de la Selva en distintas situaciones de manejo. Los parámetros estructurales muestran que las especies dominantes son Anadenanthera colubrina var cebil, Phyllostilon rhamnoides, Trichilia hieronymi, Parapiptadenia excelsa y Calycophyllum multiflorum. Las bajas tasas de incorporaciones y existencias de las especies de mayor valor, Cedrela balansae, Myroxylon peruiferum y Amburana cearensis, determinan su vulnerabilidad. La dinámica está determinada por la formación de claros que generan cambios en las condiciones de luz. Las variables que influyen sobre el crecimiento diamétrico son el tamaño del individuo, la posición sociológica y la densidad del rodal. Las especies de mayores crecimientos diamétricos son C. balansae, A. colubrina var. cebil, P. excelsa, Astronium urundeuva y M. peruiferum siendo el tiempo estimado en lograr sus diámetros mínimos de corta (DMC)de 50, 35, 60, 55 y 80 años respectivamente. La reducción de la densidad del rodal favorece los incrementos diamétricos de los individuos de las especies de valor forestal. La proyección del rodal demuestra que un aprovechamiento forestal realizado con las condiciones técnicas y DMC vigentes, la estructura, la dinámica y el volumen aprovechable no se recupera en un período de 25 años.

Novel bioassays to examine the host-finding ability of plant-parasitic nematodes

We present two novel bioassays to be used in the examination of plant-parasitic nematode host-finding ability. The host-finding 'pipette-bulb assay' was constructed from modelled Pasteur pipette bulbs and connecting barrels using parafilm fastenings. This assay examines the direction of second-stage juvenile (J2) migration in response to a host seedling, through a moistened sand substrate, which underlies terminal upward-facing 'seedling bulbs', one containing a host seedling in potting compost, the other with only potting compost. An equal watering regime through both upward-facing seedling bulbs creates a directional concentration gradient of host diffusate chemotactic factors. Positive chemotactic stimuli cause the J2 to orientate and migrate towards the host plant. We present validation data collected from assays of the root-knot nematode, Meloidogyne incognita, and the potato cyst nematode, Globodera pallida, which indicate a highly significant positive attraction of J2 of both species to respective host plants. This represents a simple, quick and inexpensive method of assessing host-finding behaviour in the laboratory. We consider that the pipette-bulb assay improves on previous host-finding/chemo-attraction assays through creating a more biologically relevant environment for experimental J2; analysis is quick and easy, allowing the straightforward interpretation of results. In addition, we have developed an 'agar trough' sensory assay variant which we believe can be used rapidly to ratify nematode responses to chemical gustatory or olfactory cues. This was constructed from a water agar substrate such that two counting wells were connected by a raised central trough, all flooded with water. Two small water agar plugs were dehydrated briefly in an oven and then hydrated in either an attractant, repellent or water control; these plugs were then placed in the terminal counting wells and subsequently leached the attractant or repellent to form a concentration gradient along the central trough, which contained the initial J2 innoculum. Our data show that both M. incognita and G. pallida J2 are positively attracted to host diffusates. In addition, they displayed a strong repulsion in response to 1 M NaCl2. J2 of M. incognita displayed a mild aversion to a non-host oak root diffusate, whereas G. pallida J2 displayed a strong aversion to the same non-host diffusate; neither species responded to a compost leachate. We believe that the agar trough assay improves on previous methods by facilitating rapid diffusion of attractant or repellents. Both of the aforementioned assays were designed as tools to assess the impact of RNAi-based reverse genetics screens for gene targets involved in chemosensory orientation.

flp-32 Ligand/Receptor Silencing Phenocopy Faster Plant Pathogenic Nematodes

Restrictions on nematicide usage underscore the need for novel control strategies for plant pathogenic nematodes such as Globodera pallida (potato cyst nematode) that impose a significant economic burden on plant cultivation activities. The nematode neuropeptide signalling system is an attractive resource for novel control targets as it plays a critical role in sensory and motor functions. The FMRFamide-like peptides (FLPs) form the largest and most diverse family of neuropeptides in invertebrates, and are structurally conserved across nematode species, highlighting the utility of the FLPergic system as a broad-spectrum control target. flp-32 is expressed widely across nematode species. This study investigates the role of flp-32 in G. pallida and shows that: (i) Gp-flp-32 encodes the peptide AMRNALVRFamide; (ii) Gp-flp-32 is expressed in the brain and ventral nerve cord of G. pallida; (iii) migration rate increases in Gp-flp-32-silenced worms; (iv) the ability of G. pallida to infect potato plant root systems is enhanced in Gp-flp-32-silenced worms; (v) a novel putative Gp-flp-32 receptor (Gp-flp-32R) is expressed in G. pallida; and, (vi) Gp-flp-32R silenced worms also display an increase in migration rate. This work demonstrates that Gp30 flp-32 plays an intrinsic role in the modulation of locomotory behaviour in G. pallida, and putatively interacts with at least one novel G-protein coupled receptor (Gp-flp-32R). This is the first functional characterisation of a parasitic nematode FLP-GPCR. © 2013 Atkinson et al.

Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens

Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants.

Trophic plasticity in the cnidarian-dinoflagellate symbiosis

Coral reefs are of utmost ecological and economical importance but are currently in global decline due to climate change and anthropogenic disturbances. Corals, as well as other cnidarian species, live in symbiosis with photosynthetic dinoflagellates of the genus Symbiodinium. This relationship provides the cnidarian host with alternative metabolic pathways, as the symbionts translocate photosynthetic carbon to the animal. Besides this autotrophic nutrition mode, symbiotic cnidarians also take up organic matter from the environment (heterotrophy). The nutritional balance between auto- and heterotrophy is critical for the functioning, fitness and resilience of the cnidariandinoflagellate symbiosis. New methodological approaches were developed to better understand the role of auto- and heterotrophy in the ecophysiology of cnidarians associated with Symbiodinium, and the ecological implications of this trophic plasticity. Specifically, the new approaches were developed to assess photophysiology, biomass production of the model organism Aiptasia sp. and molecular tools to investigate heterotrophy in the cnidarian-dinoflagellate symbiosis. Using these approaches, we were able to non-invasively assess the photophysiological spatial heterogeneity of symbiotic cnidarians and identify spatial patterns between chlorophyll fluorescence and relative content of chlorophyll a and green-fluorescent proteins. Optimal culture conditions to maximize the biomass production of Aiptasia pallida were identified, as well as their implications on the fatty acid composition of the anemones. Molecular trophic markers were used to determine prey digestion times in symbiotic cnidarians, which vary between 1-3 days depending on prey species, predator species and the feeding history of the predator. This method was also used to demonstrate that microalgae is a potential food source for symbiotic corals. By using a stable isotope approach to assess the trophic ecology of the facultative symbiotic Oculina arbuscula in situ, it was possible to demonstrate the importance of pico- and nanoplanktonic organisms, particularly autotrophic, in the nutrition of symbiotic corals. Finally, we showed the effects of functional diversity of Symbiodinium on the nutritional plasticity of the cnidarian-dinoflagellate symbiosis. Symbiont identity defines this plasticity through its individual metabolic requirements, capacity to fix carbon, quantity of translocated carbon and the host’s capacity to feed and digest prey.

In vitro degradability of mature and immature leaves of tropical forage legumes differing in condensed tannin and non-starch polysaccharide content and composition

A study was designed to examine the relationships between protein, condensed tannin and cell wall carbohydrate content and composition and the nutritional quality of seven tropical legumes (Desmodium ovalifolium, Flemingia macrophylla, Leucaena leucocephala, L pallida, L macrophylla, Calliandra calothyrsus and Clitotia fairchildiana). Among the legume species studied, D ovalifolium showed the lowest concentration of nitrogen, while L leucocephala showed the highest. Fibre (NDF) content was lowest in C calothyrsus, L Leucocephala and L pallida and highest in L macrophylla, which had no measurable condensed tannins. The highest tannin concentration was found in C calothyrsus. Total non-structural polysaccharides (NSP) varied among legumes species (lowest in C calothyrsus and highest in D ovalifolium), and glucose and uronic acids were the most abundant carbohydrate constituents in all legumes. Total NSP losses were lowest in F macrophylla and highest in L leucocephala and L pallida. Gas accumulation and acetate and propionate levels were 50% less with F macrophylla and D ovalifolium as compared with L leucocephala. The highest levels of branched-chain fatty acids were observed with non-tanniniferous legumes, and negative concentrations were observed with some of the legumes with high tannin content (D ovalifolium and F macrophylla). Linear regression analysis showed that the presence of condensed tannins was more related to a reduction of the initial rate of gas production (0-48 h) than to the final amount of gas produced or the extent (144h) of dry matter degradation, which could be due to differences in tannin chemistry. Consequently, more attention should be given in the future to elucidating the impact of tannin structure on the nutritional quality of tropical forage legumes. (C) 2003 Society of Chemical Industry.