Grazing preferences of three species of amoebae on cyanobacteria and green algae.

Twenty species/isolates of cyanobacteria and green algae were isolated from cyanobacterial bloom samples in lakes associated with the upper Qu'Appelle River drainage system in southern Saskatchewan, Canada. Three amoebae species (Cochliopodium sp., Vannella sp. and Vermamoeba vermiformis) were also isolated from one of these samples, and were subjected to grazing assays to determine which species of cyanobacteria or algae could potentially serve as a food source. Amoeba grazing rates were quantified based on the diameter of the plaque after 12 days on agar plate assays, and by estimation of the amoeba population growth rate from the rate of increase of plaque area. The common cyanobacterial bloom-formers Dolichospermum sp. and Aphanizomenon flos-aquae supported high growth rates for all three amoebae, while green algae, with the exception of one green alga/amoeba combination, did not support growth of the tested amoebae. Many of the cyanobacterial and algal isolates that did not support amoebae growth were ingested, suggesting that ingestion did not determine grazing success. Overall, while the cyanobacteria Dolichospermum sp. and Aphanizomenon flos-aquae were suitable food sources for the amoebae, the other cyanobacteria were grazed in an unpredictable manner, with some species/strains grazed by some amoebae and some species not grazed at all.

An at-home laboratory in plant biology designed to engage students in the process of science.

The COVID-19 pandemic prompted a transition to remote delivery of courses that lack immersive hands-on research experiences for undergraduate science students, resulting in a scientific research skills gap. In this report, we present an option for an inclusive and authentic, hands-on research experience that all students can perform off-campus. Biology students in a semester-long (13 weeks) sophomore plant physiology course participated in an at-home laboratory designed to study the impacts of nitrogen addition on growth rates and root nodulation by wild nitrogen-fixing Rhizobia in Pisum sativum (Pea) plants. This undergraduate research experience, piloted in the fall semester of 2020 in a class with 90 students, was created to help participants learn and practice scientific research skills during the COVID-19 pandemic. Specifically, the learning outcomes associated with this at-home research experience were: (1) generate a testable hypothesis, (2) design an experiment to test the hypothesis, (3) explain the importance of biological replication, (4) perform meaningful statistical analyses using R, and (5) compose a research paper to effectively communicate findings to a general biology audience. Students were provided with an at-home laboratory kit containing the required materials and reagents, which were chosen to be accessible and affordable in case students were unable to access our laboratory kit. Students were guided through all aspects of research, including hypothesis generation, data collection, and data analysis, with video tutorials and live virtual sessions. This at-home laboratory provided students an opportunity to practice hands-on research with the flexibility to collect and analyze their own data in a remote setting during the COVID-19 pandemic. This, or similar laboratories, could also be used as part of distance learning biology courses.

Analyzing the Biochemical Alteration of Green Algae During Chronic Exposure to Triclosan Based on Synchrotron-Based Fourier Transform Infrared Spectromicroscopy.

The study explored the chronic toxicity of triclosan to green microalga Chlorococcum sp. under multiple interactions among multiple environmental conditions. This is the first study on chronic algal toxicity to combine synchrotron-based Fourier transform infrared spectromicroscopy, factorial analysis, principal component analysis, and stepwise-cluster analysis. Such a combination helps to reveal the toxic mechanism at the molecular level and explore the inner correlationship among multiple environmental conditions. In the 120-h test, nitrogen content became the most significant factor of the physiochemical properties. Some insignificant factors in the 48-h test became significant in the 120-h test. Temperature * nitrogen content, temperature * phosphorus content, and pH * phosphorus content were the most significant two-order interactions. Temperature * pH * NaCl concentration and temperature * NaCl concentration * phosphorus content were the most significant three-order interactions. More high-order interactions became significant in the 120-h test, indicating the complexity and impacts of all the factors may increase when time was extended. The chronic toxicity of triclosan presented more distinguishable variations among treatments based on biochemical alterations. These results demonstrate that the sensitivity and fragility of algae to triclosan can be amplified with time extension. Long-term exposure can be applied to better evaluate and predict the environmental toxicity behavior of triclosan. It can also help with environmental evaluation and risk management of real-world triclosan toxicity.

Insights into Long-Term Toxicity of Triclosan to Freshwater Green Algae in Lake Erie.

This study explored the long-term impacts of a pulse disturbance of triclosan on five nontarget green algae in Lake Erie. Comprehensive analyses were performed using multiple physiological end points at community and subcellular scales. The toxic mechanism of triclosan in a wide range of concentrations was analyzed. The diverse sensitivity of algae species and complex interrelationships among multiple end points were revealed. The results showed the taxonomic groups of algae were the key issue for sensitivity difference. High doses of triclosan caused irreversible damage on algae, and environmentally relevant doses initiated either inhibition or stimulation. Smaller cells had higher sensitivity to triclosan, while larger cells had a wider size variation after exposure. Colonial cells were less sensitive than unicells. For chlorophyll, there were better dose-response relationships in Chlorococcum sp., Chlamydomonas reinhardtii CPCC 12 and 243 than Asterococcus superbus and Eremosphaera viridis. For chlorophyll fluorescence, Fv/ Fm was the most sensitive parameter, and qN was more sensitive than qP. Triclosan showed long-term effects on biochemical components, such as lipids, proteins, and nucleic acids. The findings will be helpful for a systematic and complete assessment of triclosan toxicity in natural waters and the development of appropriate strategies for its risk management.

Insights into the Toxicity of Triclosan to Green Microalga Chlorococcum sp. Using Synchrotron-Based Fourier Transform Infrared Spectromicroscopy: Biophysiological Analyses and Roles of Environmental Factors.

This study investigated the toxicity of triclosan to the green microalga Chlorococcum sp. under multiple environmental stressors. The interactions between triclosan and environmental stressors were explored through full two-way factorial, synchrotron-based Fourier transform infrared spectromicroscopy and principal component analyses. Phosphorus concentration, pH * phosphorus concentration, and temperature * pH * NaCl concentration were the most statistically significant factors under triclosan exposure. The variation of those factors would have a huge impact on biophysiological performances. It is interesting to find Chlorococcum sp. may become more resistant against triclosan in phosphorus-enriched environment. Besides, particular significant factors from multiple environmental stressors showed the impacts of triclosan on the corresponding response of Chlorococcum sp. owing to the specific structure and performance of biomolecular components. Moreover, two high-order interactions of temperature * pH * NaCl concentration and temperature * pH * NaCl concentration * phosphorus concentration had more contributions than others at the subcellular level, which could be attributed to the interactive complexity of biomolecular components. Due to cellular self-regulation mechanism and short exposure time, the biophysiological changes of Chlorococcum sp. were undramatic. These findings can help reveal the interactive complexity among triclosan and multiple environmental stressors. It is suggested that multiple environmental stressors should be considered during ecological risk assessment and management of emerging pollutants.

Molecular toxicity of triclosan and carbamazepine to green algae Chlorococcum sp.: A single cell view using synchrotron-based Fourier transform infrared spectromicroscopy.

Although pharmaceuticals and personal care products have been used and introduced into the environment in large quantities, little information on potential ecological risks is currently available considering their effects on living organisms. We verified the feasibility of using synchrotron-based Fourier Transform Infrared (SR-FTIR) spectromicroscopy to explore in vivo toxic effects on single living Chlorococcum sp. cells. The study provided important information to achieve a better understanding of the toxic mechanism of triclosan and carbamazepine on living algae Chlorococcum sp.. Triclosan and carbamazepine had distinctive toxic effects on unicellular living algae. Most strikingly, triclosan had more dramatic toxic effects on biochemical components than carbamazepine. Triclosan can affect algae primarily by inhibiting fatty acid synthesis and causing protein aggregation. The toxicity response was irreversible at higher concentration (100.000 µM), but attenuated at lower concentration (0.391 µM) as time extended. Carbamazepine can produce hydrophobic interactions to affect the phospholipid bilayer and work on specific proteins to disfunction the cell membrane. Carbamazepine-exposed cells developed a resistance while extending exposure time. This is the first demonstration from an ecological standpoint that SR-FTIR can provide an innovative approach to reveal the toxicity of emerging pollutants in aquatic environments.

Plasma membrane ferric reductase activity of iron-limited algal cells is inhibited by ferric chelators.

Iron-limited cells of the green alga Chlorella kesslerii use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Previous work has demonstrated that synthetic chelators both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen, two forms of dihydroxybenzoic acid) and also two formulations of the commonly-used herbicide N-(phoshonomethyl)glycine (glyphosate). The ferric forms of the larger siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. The smaller siderophores/siderophore precursors, 2,3- and 3,4-dihydroxybenzoic acids, did not support high rates of reductase in the ferric form but did inhibit reductase activity in the iron-free form. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric reductase activity also supported a large stimulation in the growth of iron-limited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N-(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N-(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.

Ferric and cupric reductase activities by iron-limited cells of the green alga Chlorella kessleri: quantification via oxygen electrode.

The colorimetric Fe2+ indicators bathophenanthroline disulfonic acid (BPDS) and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (FZ) are routinely used to assay for plasma membrane ferric reductase activity in iron-limited algal cells and also in roots from iron-limited plants. Ferric reductase assays using these colorimetric indicators must take into account the fact that Fe3+ chelators (e.g. ethylenediaminetetraacetic acid) can also in general bind Fe2+ and may therefore compete with the colorimetric Fe2+ indicators, leading to the potential for underestimation of the ferric reduction rate. Conversely, the presence of BPDS or FZ may also facilitate the reduction of Fe3+ chelates, potentially leading to overestimation of ferric reduction rates. Last, both BPDS and FZ have non-negligible affinities for Fe3+ in addition to their well-known affinities for Fe2+; this leads to potential difficulties in ascertaining whether free and/or chelated Fe3+ are potential substrates for the ferric reductase. Similar issues arise when assaying for cupric reductase activity using the colorimetric Cu+ indicator bathocuproinedisulfonic acid (BCDS). In this paper, we describe an oxygen-electrode-based assay (conducted in darkness) for both ferric and cupric reductase activities that does not use colorimetric indicators. Using this assay system, we show that the plasma membrane metal reductase activity of iron-limited cells of the green alga Chlorella kessleri reduced complexed Fe3+ (i.e. Fe3+ chelates) but did not reduce free (non-chelated) Fe3+, and also reduced free Cu2+ to Cu+, but did not reduce Cu2+ that was part of Cu2+ chelates. We suggest that the potential for reduction of free Fe3+ cannot be adequately assayed using colorimetric assays. As well, the BPDS-based assay system consistently yielded similar estimates of ferric reductase activity compared with the O2-electrode-based assays at relatively low Fe3+ concentration, but higher estimates at higher Fe3+ concentrations with chelators other than desferrioxamine mesylate. With respect to cupric reductase activity, the O2 electrode consistently provided much higher estimates; we suggest that this was as a result of Cu2+ chelation by BCDS leading to a large underestimation of the true cupric reduction rate. These results suggest that an O2-electrode-based metal reductase assay system has some specific advantages compared with the traditional colorimetric assay system, including especially the ability to discriminate between the reduction of free metal ions and chelated metal ions.