Tadalafil impacts the mechanical properties of Plasmodium falciparum gametocyte-infected erythrocytes.

Plasmodium falciparum gametocytes modify the mechanical properties of their erythrocyte host to persist for several weeks in the blood circulation and to be available for mosquitoes. These changes are tightly regulated by the plasmodial phosphodiesterase delta that decreases both the stiffness and the permeability of the infected host cell. Here, we address the effect of the phosphodiesterase inhibitor tadalafil on deformability and permeability of gametocyte-infected erythrocytes. We show that this inhibitor drastically increases isosmotic lysis of gametocyte-infected erythrocytes and impairs their ability to circulate in an in vitro model for splenic retention. These findings indicate that tadalafil represents a novel drug lead potentially capable of blocking malaria parasite transmission by impacting gametocyte circulation.

Survival and regeneration ability of clonal common milkweed (Asclepias syriaca L.) after a single herbicide treatment in natural open sand grasslands.

Invasive species are a major threat to biodiversity, human health, and economies worldwide. Clonal growth is a common ability of most invasive plants. The clonal common milkweed Asclepias syriaca L. is the most widespread invasive species in Pannonic sand grasslands. Despite of being an invader in disturbed semi-natural vegetation, this plant prefers agricultural fields or plantations. Herbicide treatment could be one of the most cost-effective and efficient methods for controlling the extended stands of milkweed in both agricultural and protected areas. The invasion of milkweed stand was monitored from 2011 to 2017 in a strictly protected UNESCO biosphere reserve in Hungary, and a single herbicide treatment was applied in May 2014. This single treatment was successful only in a short-term but not in a long-term period, as the number of milkweed shoots decreased following herbicide treatment. The herbicide translocation by rhizomatic roots induced the damage of dormant bud banks. The surviving buds developing shoots, growth of the milkweed stand showed a slow regeneration for a longer-term period. We concluded that the successful control of milkweed after herbicide treatment depends on repeated management of treated areas to suppress further spreading during subsequent seasons.

Polyps of the Jellyfish Aurelia aurita Are Unaffected by Chronic Exposure to a Combination of Pesticides.

Pesticides are a major contaminant in coastal waters and can cause adverse effects in marine invertebrates such as jellyfish. Most studies have investigated short-term responses of organisms to unrealistically high concentrations of pesticides; however, chronic exposure to persistent low concentrations, which are more likely to occur in the environment, are rarely analyzed. We tested the response of polyps of the moon jellyfish Aurelia aurita to environmental concentrations of the herbicide atrazine and the insecticide chlorpyrifos, individually and in combination, over 9 wk. We hypothesized that exposure to individual pesticides would reduce rates of asexual reproduction and alter polyps' metabolite profiles, and that the results would be more severe when polyps were exposed to the combined pesticides. Polyps survived and reproduced (through budding) in all treatments, and no differences among treatments were observed. Proton nuclear magnetic resonance spectroscopy revealed no difference in profiles of polar metabolites of polyps exposed to the individual or combined pesticides. Our results suggest that A. aurita polyps are unaffected by chronic exposure to atrazine and chlorpyrifos at concentrations recommended as being protective by current Australian water quality guidelines. Environ Toxicol Chem 2020;39:1685-1692. © 2020 SETAC.

Antimalarial pantothenamide metabolites target acetyl-coenzyme A biosynthesis in Plasmodium falciparum.

Malaria eradication is critically dependent on new therapeutics that target resistant Plasmodium parasites and block transmission of the disease. Here, we report that pantothenamide bioisosteres were active against blood-stage Plasmodium falciparum parasites and also blocked transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties, and cleared parasites in a humanized mouse model of P. falciparum infection. Metabolomics revealed that coenzyme A biosynthetic enzymes converted pantothenamides into coenzyme A analogs that interfered with parasite acetyl-coenzyme A anabolism. Resistant parasites generated in vitro showed mutations in acetyl-coenzyme A synthetase and acyl-coenzyme A synthetase 11. Introduction and reversion of these mutations in P. falciparum using CRISPR-Cas9 gene editing confirmed the roles of these enzymes in the sensitivity of the malaria parasites to pantothenamides. These pantothenamide compounds with a new mode of action may have potential as drugs against malaria parasites.

Ivermectin Impairs the Development of Sexual and Asexual Stages of Plasmodium falciparum In Vitro.

Ivermectin is the drug of choice for many parasitic infections, with more than one billion doses being distributed in onchocerciasis programs. The drug has been put into focus recently by the malaria community because of its potential to kill blood-sucking mosquitoes, thereby reducing malaria transmission. However, the activity of ivermectin against the malaria parasite itself has been only partly investigated. This study aimed to investigate the in vitro activity of ivermectin against asexual and sexual stages of Plasmodium falciparum Both asexual and late-stage gametocytes were incubated with ivermectin and control drugs in vitro The growth-inhibiting effects were assessed for asexual stages of different Plasmodium falciparum laboratory strains and culture-adapted clinical isolates using the histidine-rich protein 2 enzyme-linked immunosorbent assay technique. The effect against stage IV/V gametocytes was evaluated based on ATP quantification. Ivermectin showed activities at nanomolar concentrations against asexual stages (50% inhibitory concentration of ∼100 nM) and stage IV/V gametocytes (500 nM) of P. falciparum Stage-specific assays suggested that ivermectin arrests the parasite cycle at the trophozoite stage. Ivermectin might add a feature to its "wonder drug" properties with activity against asexual stages of the malaria parasite Plasmodium falciparum The observed activities might be difficult to reach with current regimens but will be more relevant with future high-dose regimens under investigation. Further studies should be performed to confirm these results in vitro and in vivo.

Toxicity of manganese to various life stages of selected marine cnidarian species.

Manganese (Mn) pollution in marine waters is increasing and sensitivities to this metal vary widely among marine species. The aims of this study were to characterise Mn chemistry in seawater, and evaluate the toxic effects of Mn on various life stages of two scleractinian corals - the branching sp. Acropora spathulata and massive sp. Platygyra daedalea, and the anemone Exaiptasia pallida. Analytical and theoretical characterisation experiments showed that 97-100% of Mn (II) additions ≤ 200 mg/L in seawater were soluble over 72 h and largely assumed labile complexes. Concentrations estimated to reduce coral fertilisation success by 50% (5.5-h EC50) were 237 mg/L for A. spathulata and 164 mg/L for P. daedalea. A relatively low 72-h LC50 of 7 mg/L was calculated for A. spathulata larvae. In a pilot test using fragments of adult A. spathulata, intact coral tissue rapidly sloughed away from the underlying skeleton at very low concentrations with a 48-h EC50 of just 0.7 mg/L. For E. pallida, survival, tentacle retraction and reproduction were unaffected by prolonged high exposures (12-d NOEC 54 mg/L). This study provides important data supporting the derivation of separate water quality guidelines for Mn in systems with and without coral - a decision recently considered by Australian and New Zealand authorities. It demonstrates the high sensitivity of coral larvae and adult colonies to Mn and the potential risks associated with relying on other early life stage tests and/or E. pallida as ecotoxicological representatives of critically important scleractinian corals.

The novel fungicide SYP-14288 acts as an uncoupler against Phytophthora capsici.

SYP-14288 is a novel fungicide developed by the Shenyang Research Institute of Chemical Industry in China. Although preliminary studies indicate that SYP-14288 is highly effective against 32 important plant pathogens belonging to a range of taxonomic groups, its mode of action remains unknown. In this study, we documented that SYP-14288 has excellent activity against all of the asexual life stages of the plant-pathogenic oomycete Phytophthora capsici, and is especially effective in blocking cyst germination and other life stages that require high energy consumption. In assays designed to determine the fungicide's mode of action, addition of ATP reduced SYP-14288 inhibition of P. capsici, which suggested that SYP-14288 inhibits ATP synthesis of the pathogen. This inference was confirmed in that treatment with SYP-14288 sharply reduced the ATP content in P. capsici. The respiration rate of P. capsici was positively correlated with the concentration of SYP-14288 or of the fungicide fluazinam (an uncoupler of oxidative phosphorylation), but increases in respiration were greater with SYP-14288 than with fluazinam. These results indicate that SYP-14288 is a promising fungicide that functions as an uncoupler of oxidative phosphorylation.

Alpha-Linolenic Acid, but Not Palmitic Acid, Negatively Impacts Survival, Asexual Reproductive Rate, and Clonal Offspring Size in Hydra oligactis.

Hydra, as sit-and-wait predators with limited food selectivity, could serve as model organisms for the analysis of the effect of a particular dietary component on growth and reproduction. We investigated the effect of food quality and of diets enriched with palmitic (PAM) or α-linolenic acid (ALA) on the life history traits of two hydra species: Hydra oligactis and Hydra vulgaris. We tested the hypothesis that a diet enriched with polyunsaturated fatty acids (PUFA) can stimulate growth and reproduction in simple metazoans with a sit-and-wait type of predatory strategy. Our results revealed that a diet based on Artemia nauplii, which are not a natural food for freshwater hydra, stimulated growth, asexual reproduction, and survival in hydra. Artemia nauplii were characterized by the highest lipid content of all used food sources. The analysis of the fatty acid content of hydra indicated the domination the n-6 fatty acids over n-3 (eicosapentaenoic acid [EPA], docosahexaenoic acid [DHA], and ALA). Arachidonic acid appeared to be the dominant PUFA in Hydra, irrespective of diet supplementation with palmitic acid or ALA. The dietary supplementation of ALA negatively affected the survival, asexual reproductive rate, and size of clonal offspring of H. oligactis and had no effect on the life history traits of H. vulgaris. Our results also suggest that the hydras are not able to efficiently convert ALA into other essential fatty acids, such as EPA and DHA. To our knowledge, this is the first report about the adverse effects of n-3 fatty acid supplementation in primitive metazoans such as hydra.

Identification of an Asexual Reproduction Inducer of Phytopathogenic and Toxigenic Fusarium.

Asexual and sexual reproduction are the most important biological events in the life cycle of phytopathogenic and toxigenic Fusarium and are responsible for disease epidemics. However, the signaling molecules which induce the asexual reproduction of Fusarium are unknown. Herein we describe the structure elucidation, including the absolute configuration, of Fusarium asexual reproduction inducer (FARI), a new sesquiterpene derivative, by spectroscopic analysis, total synthesis, and conidium-inducing assays of synthetic isomers. We have also uncovered the universality of FARI among Fusarium species. Moreover, a mechanism-of-action study suggested that the Gpmk1 and LaeA signaling pathways are required for conidium formation induced by FARI; conversely, the Mgv1 of mitogen-activated protein kinase is not involved in conidium formation. FARI exhibited conidium-inducing activity at an extremely low dose and high stereoselectivity, which may suggest the presence of a stereospecific target.

A cryptic cycle in haematopoietic niches promotes initiation of malaria transmission and evasion of chemotherapy.

Blood stage human malaria parasites may exploit erythropoietic tissue niches and colonise erythroid progenitors; however, the precise influence of the erythropoietic environment on fundamental parasite biology remains unknown. Here we use quantitative approaches to enumerate Plasmodium infected erythropoietic precursor cells using an in vivo rodent model of Plasmodium berghei. We show that parasitised early reticulocytes (ER) in the major sites of haematopoiesis establish a cryptic asexual cycle. Moreover, this cycle is characterised by early preferential commitment to gametocytogenesis, which occurs in sufficient numbers to generate almost all of the initial population of circulating, mature gametocytes. In addition, we show that P. berghei is less sensitive to artemisinin in splenic ER than in blood, which suggests that haematopoietic tissues may enable origins of recrudescent infection and emerging resistance to antimalarials. Continuous propagation in these sites may also provide a mechanism for continuous transmission and infection in malaria endemic regions.

Lycosides, Unusual Carotenoid-Derived Terpenoid Glycosides from a Vegetable Juice, Inhibit Asexual Reproduction of the Plant Pathogen Phytophthora.

Vegetable juices, typical culture media for the plant pathogen Phytophthora, effectively induce its asexual reproduction (zoosporangia formation). However, some chromatographic fractions from a vegetable juice were found to inhibit asexual reproduction. Bioassay-guided chromatographic steps led to the isolation of four novel compounds, named lycosides A-D, 1-4, that could be metabolic products from a carotenoid. They showed 50% inhibitory activity against the asexual reproduction of P. capsici at 2.1-7.6 µM. The structure-activity relationship and the universality of the inhibitory activity within the Phytophthora genus were also investigated. In addition, the quantitative analysis of lycosides in fresh vegetables and vegetable juices revealed that tomato is the source of these active substances. These food-derived chemicals could help provide safe agents to control the outbreak of the agricultural pest Phytophthora in fields.

Involvement of a velvet protein ClVelB in the regulation of vegetative differentiation, oxidative stress response, secondary metabolism, and virulence in Curvularia lunata.

The ortholog of Aspergillus nidulans VelB, which is known as ClVelB, was studied to gain a broader insight into the functions of a velvet protein in Curvularia lunata. With the expected common and specific functions of ClVelB, the deletion of clvelB results in similar though not identical phenotypes. The pathogenicity assays revealed that ΔClVelB was impaired in colonizing the host tissue, which corresponds to the finding that ClVelB controls the production of conidia and the methyl 5-(hydroxymethyl) furan-2-carboxylate toxin in C. lunata. However, the deletion of clvelB led to the increase in aerial hyphae and melanin formation. In addition, ΔClVelB showed a decreased sensitivity to iprodione and fludioxonil fungicides and a decreased resistance to cell wall-damaging agents and osmotic stress and tolerance to H2O2. The ultrastructural analysis indicated that the cell wall of ΔClVelB became thinner, which agrees with the finding that the accumulated level of glycerol in ΔClVelB is lower than the wild-type. Furthermore, the interaction of ClVelB with ClVeA and ClVosA was identified in the present research through the yeast two-hybrid and bimolecular fluorescence complementation assays. Results indicate that ClVelB plays a vital role in the regulation of various cellular processes in C. lunata.

Assessing the chronic toxicity of copper and aluminium to the tropical sea anemone Exaiptasia pallida.

The world's most productive bauxite mines and alumina refineries are located in tropical or sub-tropical regions. The discharge water from alumina refineries can contain elevated aluminium (Al, <0.45µm fraction), from 30 to 1000µg/L. There is a need for additional information on the toxicity of Al to aquatic organisms to improve the environmental regulation and management of alumina refinery operations in tropical coastal regions. A 14-d chronic toxicity test was developed for the tropical sea anemone Exaiptasia pallida. Asexual reproduction and growth rates of E. pallida were assessed using the number of lacerates produced and oral disc diameter. The comparative sensitivity of E. pallida was assessed through exposure to a commonly-used reference toxicant, copper (Cu) at 28°C, with asexual reproduction toxicity estimates of 10% (EC10) and 50% (EC50) effect concentrations, calculated as 8.8µg/L (95% confidence limits (CL): 1-18µg/L) and 35µg/L Cu (95% CL: 30-39µg/L), respectively. Growth rate was a suitable additional endpoint (EC50=35µg/L Cu, 95% CL: 23-49µg/L). The EC10 and EC50 for Al (total fraction, based on reproduction) at 28°C were 817µg/L (95% CL: 440-1480µg/L) and 2270µg/L (95% CL: 1600-3900µg/L), respectively. The toxicity of Cu and Al was also assessed at 24°C and 31°C, representing average year-round water temperatures for sub-tropical and tropical Australian coastal environments. Changing the temperature from 28°C to 24°C or 31°C resulted in up to 45% less reproduction of anemones and increased their sensitivity to Cu (EC50s at 24°C=21µg/L, 95% CL: 17-26µg/L and at 31°C=23µg/L, 95% CL: 21-25µg/L). Sensitivity to Al was reduced at 24°C with an EC50 of 8870µg/L (95% CL: 6200-NC). An EC50 for Al at 31°C could not be calculated. This test is a reliable and sensitive addition to the suite of standardised tests currently developed for tropical marine species.

Competition, salinity, and clonal growth in native and introduced irises.

PREMISE OF THE STUDY: Iris pseudacorus spread rapidly into North America after introduction from Europe in the 1800s and now co-occurs with native I. hexagona in freshwater Louisiana wetlands. Native irises support and interact with multiple trophic levels, whereas I. pseudacorus is classified an invasive pest because it grows aggressively, reduces biodiversity, and displaces native vegetation. Salinity levels are increasing in coastal wetlands worldwide. We examined how salt-stress affects competitive interactions between these conspecifics. METHODS: We established a three-way full-factorial common-garden experiment that included species (I. pseudacorus, I. hexagona), competition (no competition, intraspecific competition, and interspecific competition), and salinity (0, 4, 8 parts per thousand NaCl), with six replicates per treatment. KEY RESULTS: After 18 mo, Iris pseudacorus produced much more biomass than the native species did (F1, 92 = 71.5, P < 0.0001). Interspecific competition did not affect the introduced iris, but biomass of the native was strongly reduced (competition × species interaction: F2, 95 = 76.7, P = 0.002). Salinity significantly reduced biomass of both species (F2, 92 = 21.8, P < 0.0001), with no species × salinity interaction (F2, 84 = 1.85, P = 0.16). CONCLUSIONS: Our results demonstrate that salt stress strongly reduced clonal reproduction in native and introduced irises; however, the introduced iris had a competitive advantage over the native, regardless of environmental salinity levels. Based on patterns in clonal reproduction, the introduced iris could potentially threaten native iris populations. We are currently investigating seed production and mortality during competition and stress because both clonal and sexual reproduction must be considered when predicting long-term population dynamics.

The Novel Oomycide Oxathiapiprolin Inhibits All Stages in the Asexual Life Cycle of Pseudoperonospora cubensis - Causal Agent of Cucurbit Downy Mildew.

Oxathiapiprolin is a new oomycide (piperidinyl thiazole isoxazoline class) discovered by DuPont which controls diseases caused by oomycete plant pathogens. It binds in the oxysterol-binding protein domain of Oomycetes. Growth chambers studies with detached leaves and potted plants showed remarkable activity of oxathiapiprolin against Pseudoperonospora cubensis in cucurbits. The compound affected all stages in the asexual life cycle of the pathogen. It inhibited zoospore release, cystospore germination, lesion formation, lesion expansion, sporangiophore development and sporangial production. When applied to the foliage as a preventive spray no lesions developed due to inhibition of zoospore release and cystospore germination, and when applied curatively, at one or two days after inoculation, small restricted lesions developed but no sporulation occurred. When applied later to mature lesions, sporulation was strongly inhibited. Oxathiapiprolin suppressed sporulation of P. cubensis in naturally-infected leaves. It exhibited trans-laminar activity, translocated acropetaly from older to younger leaves, and moved from the root system to the foliage. Seed coating was highly effective in protecting the developed cucumber plants against downy mildew. UV microscopy observations made with cucumber leaves infected with P. cubensis revealed that inhibition of mycelium growth and sporulation induced by oxathiapiprolin was associated with callose encasement of the haustoria.

Asexual sporulation facilitates adaptation: The emergence of azole resistance in Aspergillus fumigatus.

Understanding the occurrence and spread of azole resistance in Aspergillus fumigatus is crucial for public health. It has been hypothesized that asexual sporulation, which is abundant in nature, is essential for phenotypic expression of azole resistance mutations in A. fumigatus facilitating subsequent spread through natural selection. Furthermore, the disease aspergilloma is associated with asexual sporulation within the lungs of patients and the emergence of azole resistance. This study assessed the evolutionary advantage of asexual sporulation by growing the fungus under pressure of one of five different azole fungicides over seven weeks and by comparing the rate of adaptation between scenarios of culturing with and without asexual sporulation. Results unequivocally show that asexual sporulation facilitates adaptation. This can be explained by the combination of more effective selection because of the transition from a multicellular to a unicellular stage, and by increased mutation supply due to the production of spores, which involves numerous mitotic divisions. Insights from this study are essential to unravel the resistance mechanisms of sporulating pathogens to chemical compounds and disease agents in general, and for designing strategies that prevent or overcome the emerging threat of azole resistance in particular.

Toxicity of noradrenaline, a novel anti-biofouling component, to two non-target zooplankton species, Daphnia magna and Ceriodaphnia dubia.

Noradrenaline (NA) is the active component of novel antifouling agents and acts by preventing attachment of fouling organisms. The goal of this study was to examine the toxicity of NA to the non-target zooplankton D. magna and C. dubia. Neonates were exposed to one of five concentrations of NA and effects on survival, reproduction and molting were determined. Calculated LC50 values were determined to be 46 and 38 µM in C. dubia and D. magna, respectively. A 10-day C. dubia study found that reproduction metrics were significantly impacted at non-lethal concentrations. In D. magna, concentrations greater than 40 µM significantly impacted molting. A toxicity test was conducted with D. magna using oxidized NA, which yielded similar results. These data indicate that both NA and oxidized NA are toxic to non-target zooplankton. Results obtained from this study can be used to guide future ecological risk assessments of catecholamine-based antifouling agents.

Morphological alterations in the freshwater rotifer Brachionus calyciflorus Pallas 1766 (Rotifera: Monogononta) caused by vinclozolin chronic exposure.

Vinclozolin (VZ) is a dicarboximide fungicide widely used on fruits, vegetables and wines, effective against fungi plagues. In this study we characterized the effects of VZ using a 4-day reproductive chronic assay with the freshwater rotifer Brachionus calyciflorus. The assay included observations of several features of asexual and sexual reproduction. Our results indicate that VZ: (a) increased asexual and sexual reproduction, (b) caused severe abnormality in females and (c) these abnormalities were inherited by sexual and asexual reproduction. At 1.2 mg/L three abnormal females were found out of 457 total females (0.66 %). This low percentage is consistent and reproducible according to further analysis, where we increased the number of replicates and total females exposed to 1.2 mg/L of VZ, and found 18 abnormal females out of 2868 total females (0.63 % abnormality). Interestingly, abnormal females found at 5.6 mg/L VZ exposure, were able to show mating behavior. Our results suggest that VZ behaves as a strong endocrine disruptor whose effects show the characteristic inverted-U-shape exposure concentration response curve regarding the intrinsic population increase and the percentage of abnormalities as endpoints.

Establishment of a new method to quantitatively evaluate hyphal fusion ability in Aspergillus oryzae.

Hyphal fusion is involved in the formation of an interconnected colony in filamentous fungi, and it is the first process in sexual/parasexual reproduction. However, it was difficult to evaluate hyphal fusion efficiency due to the low frequency in Aspergillus oryzae in spite of its industrial significance. Here, we established a method to quantitatively evaluate the hyphal fusion ability of A. oryzae with mixed culture of two different auxotrophic strains, where the ratio of heterokaryotic conidia growing without the auxotrophic requirements reflects the hyphal fusion efficiency. By employing this method, it was demonstrated that AoSO and AoFus3 are required for hyphal fusion, and that hyphal fusion efficiency of A. oryzae was increased by depleting nitrogen source, including large amounts of carbon source, and adjusting pH to 7.0.

Effects of Cd, Co, Cu, Ni and Zn on asexual reproduction and early development of the tropical sea anemone Aiptasia pulchella.

Currently few studies present sub-lethal toxicity data for tropical marine species, and there are no routine toxicity tests using marine cnidarians. The symbiotic sea anemone Aiptasia pulchella has been identified as a useful species for ecotoxicological risk assessment, and would provide a tropical marine cnidarian representative. Chronic sub-lethal toxicity tests assessing the effects of 28-day trace metal exposure on asexual reproduction in A. pulchella were investigated, and concentration-dependant reductions in the number of offspring that were produced were evident for all metal exposures. Metal concentration estimates causing 50% reductions in the numbers of asexually-reproduced juveniles after 28-day exposures (28-day effect concentrations 50%: EC50s) were 14 µg/L for copper, 63 µg/L for zinc, 107 µg/L for cobalt, 145 µg/L for cadmium, and 369 µg/L for nickel. Slightly higher 28-day EC50s of 16 µg/L for copper, 192 µg/L for zinc, 172 µg/L for cobalt, 185 µg/L for cadmium, and 404 µg/L for nickel exposures and were estimated based on reductions in the total number of live developed and undeveloped offspring. These sensitive and chronic sub-lethal toxicity estimates help fill the knowledge gap related to metal effects on cnidarians over longer exposure periods, and this newly-developed bioassay may provide a much needed tool for ecotoxicological risk assessment relevant to tropical marine environments.