Community level impacts of invasive mosquitofish may exacerbate the impact to a threatened amphibian

Invasive fish threaten many native freshwater fauna. However, it can be difficult to determine how invasive fish impact animals with complex life cycles as interaction may be driven by either predation of aquatic larvae or avoidance of fish‐occupied waterbodies by the terrestrial adult stage. Mosquitofish (Gambusia spp.) are highly successful and aggressive invaders that negatively impact numerous aquatic fauna. One species potentially threatened by Gambusia holbrooki is the green and golden bell frog (Litoria aurea). However, G. holbrooki's role in this frog's decline was unclear due to declines driven by the chytrid fungal disease and the continued co‐existence of these fish and frogs in multiple locations. To clarify the extent to which Gambusia is impacting L. aurea, we conducted 3 years of field surveys across a deltaic wetland system in south‐east Australia. We measured the presence and abundance of aquatic taxa including G. holbrooki, and L. aurea frogs and tadpoles, along with habitat parameters at the landscape and microhabitat scale. Generalized linear models were used to explore patterns in the abundance and distributions of L. aurea and G. holbrooki. We found strong negative associations between G. holbrooki and tadpoles of most species, including L. aurea, but no apparent avoidance of G. holbrooki by adult frogs. Native invertebrate predators (Odonata and Coleoptera) were also absent from G. holbrooki‐occupied ponds. Due to the apparent naivety of adult frogs toward G. holbrooki, the separation of G. holbrooki and tadpoles, plus the abundance of alternative predators in G. holbrooki‐free ponds, we conclude that the impact of G. holbrooki on L. aurea recruitment is likely substantial and warrants management action.     

The effects of microbial exopolysaccharides (EPS) in vase water on the water relations and the vase life of Rosa cv. Sonia

Pure cultures of five microbial species were used to test the formation of exopolysaccharides (EPS) when grown in agitated sucrose (5% w/v) containing liquid cultures. These test species were isolated from stems of freshly harvested cut flowers ( Chrysanthemum, Gerbera and Rosa ) or from the vase water of these flower cultivars. The partial conversion of sucrose into other saccharides was demonstrated by HPLC and colorimetric analysis. The final polymeric character of the newly formed saccharides was investigated. SEM preparations of xylem vessels of Rosa maintained in EPS-containing vase water showed blockage, disorganization and injury of the vessel structure. EPS were shown not to pass the xylem pit membranes. Recovery from the first symptoms of disturbed water flow (wilting) due to EPS was possible in young flowers by cutting off the blocked part of the stem (15–20 cm. The higher the microbial conversion rate of sucrose into polysaccharides, the more disturbed were the water relations of the roses placed in the EPS-containing fluid, as was demonstrated by the decrease of: (1) water conductivity of Rosa stem segments (ml/30 min); (2) water uptake (ml/d); (3) Rosa vase life (d); and (4) flower bud development. Bacterial EPS (presumably levans and dextrans) could be concentrated in the retentate by molecular filtration with a cut-off level of 10000 Da. Filtrates did not cause Rosa xylem blockage and 'bent-neck'of the flower stems, but still may be toxic to roses. Two simple methods were also used for diagnostic investigations: (1) the beetroot tissue cube test to detect microbial products causing injury of the plant cell membranes, (2) the acid fuchsin test, to show the extent and location of Rosa xylem vessel occlusion.     

A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel 'gene insertion/marker out' (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research.     

Structure-based lead identification of ATP-competitive MK2 inhibitors

MK2 kinase is a promising drug discovery target for the treatment of inflammatory diseases. Here, we describe the discovery of novel MK2 inhibitors using X-ray crystallography and structure-based drug design. The lead has in vivo efficacy in a short-term preclinical model.     

Ultra High Content Image Analysis and Phenotype Profiling of 3D Cultured Micro-Tissues

In many situations, 3D cell cultures mimic the natural organization of tissues more closely than 2D cultures. Conventional methods for phenotyping such 3D cultures use either single or multiple simple parameters based on morphology and fluorescence staining intensity. However, due to their simplicity many details are not taken into account which limits system-level study of phenotype characteristics. Here, we have developed a new image analysis platform to automatically profile 3D cell phenotypes with 598 parameters including morphology, topology, and texture parameters such as wavelet and image moments. As proof of concept, we analyzed mouse breast cancer cells (4T1 cells) in a 384-well plate format following exposure to a diverse set of compounds at different concentrations. The result showed concentration dependent phenotypic trajectories for different biologically active compounds that could be used to classify compounds based on their biological target. To demonstrate the wider applicability of our method, we analyzed the phenotypes of a collection of 44 human breast cancer cell lines cultured in 3D and showed that our method correctly distinguished basal-A, basal-B, luminal and ERBB2+ cell lines in a supervised nearest neighbor classification method.     

Diversity and species composition of West African ungulate assemblages: effects of fire,climate and soil

Aim Anthropogenic fires are a major component of the ecology of rangelands throughout the world. To assess the effects of these fires on the diversity patterns of herbivores, we related gradients in fire occurrence, climate and soil fertility to patterns in alpha and beta diversity of African ungulates. Location West Africa. Methods We used a survey‐based approach for ungulates in 37 protected areas in desert, savanna and rain forest habitats throughout West Africa, combined with satellite images of fire occurrence and digital maps of actual evapotranspiration and soil fertility. Alpha diversity was related to the environmental variables using conventional and spatial regression models. We investigated beta diversity using partial Mantel tests and ordination techniques, and by partitioning the variance in assemblage composition into environmental and spatial components. Results The species richness of grazers showed a quadratic relationship with actual evapotranspiration, whereas that of browsers and frugivores showed a linear relationship. However, in the multiple regression models fire occurrence was the only variable that significantly correlated with the species richness of grazers. Soil fertility was weakly related to overall beta diversity and the species richness of browsers, but was non‐significant in the multiple regression models. Fire occurrence was the most important variable explaining species composition of the overall species set and of grazers, whereas the assemblage composition of browsers and frugivores was explained mostly by actual evapotranspiration. Main conclusions In contrast to previous studies, our analyses show that moisture and nutrients alone fail to adequately predict the diversity patterns of grazing ungulates. Rather, the species richness and assemblage composition of grazers are largely governed by anthropogenic fires that modify the quality and structure of the grass sward. Diversity patterns of browsers and frugivores are markedly different from grazers and depend mainly on the availability of moisture, which is positively correlated with the availability of foliage and fruits. Our study highlights the importance of incorporating major human‐induced disturbances or habitat alterations into analyses of diversity patterns.     

A new population of Bleeding-heart Pigeon (Gallicolumba sp.) and its conservation relevance on Panay, Philippines

Summary Here we report on a newly discovered population of Bleeding-heart Pigeon (Gallicolumba sp.) on the Philippine island of Panay, West Visayas. There have been 7+ sight-records and one photographic documentation in two places (Lahang, Sibaliw) of the low-elevation forest of the northwest Panay peninsula (400 – 450 m a.s.l.) in 1997. Reports from local hunters received in 1995 point to the Bleeding-heart's occurrence also in the larger Panay mountain range in the west of the island. These latter records could not be substantiated during altogether 6.5 months of field work at low-elevation (Alujipan, 450 m a.s.l.) and high-elevation (Hamtang forest, Mt Balabag, 900 – 950 m a.s.l.) sites in 1995 and 1996. The photograph obtained indicates the new population to belong toGallicolumba keayi of neighbouring Negros island. — For unknown reasons the new Bleeding-heart must be extremely rare. Its still unsettled taxonomic position together with its rarity, give reasons for most serious conservation concern.

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Eine neue Population der Dolchstichtaube (Gallicolumba spec.) und ihr Arterhaltungsstatus auf Panay, Philippinen

Zusammenfassung Im Zug systematischer Erfassung der zoologisch wenig erforschten Insel Panay, Philippinen, wurde 1997 im Submontanwald der NW-Halbinsel eine bisher unbekannte neue Dolchstichtaube (Gallicolumba spec.) entdeckt. Diese nach Sichtbeobachtungen und einem Foto der auf Negros lebenden Art (G. keayi) nahestehende Form ist äußerst selten. Die noch unklare systematische Stellung und die Seltenheit geben Anlaß zu größter Besorgnis hinsichtlich der Erhaltung der neuen Form.

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This is publication No. 12 of the Philippine Endemic Species Conservation Project (PESCP).     

A metabolic model of the biological phosphorus removal process: I. Effect of the sludge retention time

The biological phosphorus removal process is a process which depends basically on three internal storage compounds. Poly-beta-hydroxybutyrate (PHB) produced during the anaerobic phase is used as substrate for biomass, polyphosphate, and glycogen formation. The reaction rates of the aerobic processes are primarily determined by the PHB content of the cells. This PHB content is highly dynamic due to the conversions during the anaerobic and aerobic phase of the cycle and the ratio between substrate addition and biomass present in the reactor. The amount of biomass present in the reactor is determined by the sludge retention time and growth rate. A metabolic model of the biological phosphorus removal process was developed and verified over a wide range of growth rates. The effect of different growth rates on the internal fractions of stored components was determined and described mathematically. One set of kinetic parameters was capable of describing the measured conversions of all components observed in the reactor as a function of the sludge retention time. (c) 1995 John Wiley & Sons, Inc.     

Proteomic and Genetic Analyses Demonstrate that Plasmodium berghei Blood Stages Export a Large and Diverse Repertoire of Proteins

Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.Malaria parasites invade and develop inside red blood cells, and extensive remodeling of the host cell is required in order for the parasite to take up nutrients and grow (1). In addition, infected red blood cells (irbcs)¹ of several Plasmodium species adhere to endothelium lining blood capillaries, and this is achieved through modification of the irbc, specifically, alteration of the irbc membrane (2, 3). This active remodeling of the erythrocyte requires the export of parasite proteins into the host cell cytoplasm and their incorporation in the irbc membrane of the host cell (1, 2). Schizont-infected red blood cells of the rodent parasite P. berghei ANKA adhere to endothelial cells of the microvasculature, leading to the sequestration of irbcs in organs such as the lungs and adipose tissue (4–6). P. berghei irbcs adhere to the class II scavenger receptor CD36 (7), which is highly conserved in mammals and is the receptor most commonly used by irbcs infected with the human parasite P. falciparum (8). These observations suggest that P. berghei may export proteins onto the surface of irbcs in a fashion analogous to the processes employed by P. falciparum that expresses PfEMP1, the protein known to be responsible for P. falciparum irbc sequestration. However, P. berghei does not contain Pfemp1 orthologs or proteins with domains with clear homology to the domains of PfEMP1 (9), and the P. berghei proteins responsible for irbc cytoadherence and proteins involved in the transport of these proteins to the irbc membrane remain largely unknown. Recently we used a proteomic analysis of P. berghei ANKA irbc membranes to identify parasite proteins associated with the erythrocyte membrane, and we have demonstrated that the deletion of a single-copy gene of P. berghei that encodes a small exported protein known as SMAC results in strongly reduced irbc sequestration (6). No evidence was found for the presence of SMAC on the irbc surface, and therefore this protein is most likely involved in the transport or anchoring of other P. berghei proteins that directly interact with host receptors on endothelial cells.For P. falciparum, a large number of exported proteins have been predicted based on the presence of an N-terminal motif known as the Plasmodium export element (PEXEL) motif (10, 11). Many of these PEXEL-positive proteins belong to species-specific gene families. Comparison of PEXEL-positive proteins in different Plasmodium species suggested that P. falciparum expresses a significantly higher number of exported proteins than other Plasmodium species, which in part can be attributed to the expansion of P. falciparum–specific protein families, including those containing DnaJ or PHIST domains (12–17). One explanation for the elevated number of exported proteins in P. falciparum is that they are necessary for export of the P. falciparum–specific protein PfEMP1 to the irbc surface (10). Comparisons of different Plasmodium exportomes have mainly focused on identifying orthologs of the PEXEL-positive proteins of P. falciparum in the other species (14, 15, 18). For example, of the >500 PEXEL-positive P. falciparum proteins, only between 11 and 33 had orthologs in P. berghei (14, 15, 19). However, such an approach might underestimate the total number of exported proteins. A recent hidden Markov model (HMM) analysis of the PEXEL motif for P. berghei proteins identified at least 75 PEXEL-positive P. berghei proteins (6). Moreover, in different Plasmodium species, a number of exported proteins have been described that are PEXEL-negative, indicating that alternative export pathways might exist that are independent of the presence of a PEXEL motif (20, 21). It has been suggested that in species with a small number of PEXEL-positive proteins, PEXEL-negative exported proteins play a more prominent role in host cell remodeling (21). An example of a PEXEL-negative exported protein family is the large PIR family of proteins, which are expressed by rodent Plasmodium species (9, 22), the monkey parasite P. knowlesi (23), and the human parasite P. vivax (24, 25).To date, export to the irbc cytosol has been shown for only a few P. berghei proteins (i.e. several members of the BIR family; TIGR01590) (6), two members of the ETRAMP family (26), and two proteins encoded by a single copy gene, SMAC and IBIS1 (6, 27). In this study, comparative proteomic, genomic, and reverse-genetic approaches have been used to identify novel exported proteins of P. berghei. We report proteome analyses of samples enriched for proteins associated with membranes of irbcs from both sequestering P. berghei ANKA and non-sequestering P. berghei K173 parasites, and we also present analyses of the full genome sequence of a non-sequestering P. berghei K173 line. Fluorescent tagging of parasite proteins selected from the proteome and genome analyses identified a number of novel P. berghei ANKA proteins that are exported into the irbc cytoplasm. We report for the first time the export of members of the PEXEL-negative Pb-fam-1 gene family (pyst-a; TIGR01599) and show that two proteins are transported to the P. berghei ANKA irbc membrane. This is the first comprehensive study of exported proteins of P. berghei that has been validated via the generation of a large number of transgenic P. berghei ANKA parasites expressing tagged proteins and has shown the export of both PEXEL-positive and PEXEL-negative proteins to the irbc cytoplasm. The identification of P. berghei ANKA proteins exported to the irbc membrane and proteins involved in sequestration suggests the possibility of developing “humanized” small animal models for the in vivo analysis of the sequestration properties of P. falciparum proteins that would express (domains of) P. falciparum proteins on the surface of rodent irbcs (4, 6).