A reply to "Relevant factors in the eutrophication of the Uruguay River and the Río Negro".

A recent paper by Beretta-Blanco and Carrasco-Letelier (2021) claims that agricultural eutrophication is not one of the main causes for cyanobacterial blooms in rivers and artificial reservoirs. By combining rivers of markedly different hydrological characteristics e.g., presence/absence and number of dams, river discharge and geological setting, the study speculates about the role of nutrients for modulating phytoplankton chlorophyll-a. Here, we identified serious flaws, from erratic and inaccurate data manipulation. The study did not define how erroneous original dataset values were treated, how the variables below the detection/quantification limit were numerically introduced, lack of mandatory variables for river studies such as flow and rainfall, arbitrary removal of pH > 7.5 values (which were not outliers), and finally how extreme values of other environmental variables were included. In addition, we identified conceptual and procedural mistakes such as biased construction/evaluation of model prediction capability. The study trained the model using pooled data from a short restricted lotic section of the (large) Uruguay River and from both lotic and reservoir domains of the Negro River, but then tested predictability within the (small) Cuareim River. Besides these methodological considerations, the article shows misinterpretations of the statistical correlation of cause and effect neglecting basic limnological knowledge of the ecology of harmful algal blooms (HABs) and international research on land use effects on freshwater quality. The argument that pH is a predictor variable for HABs neglects overwhelming basic paradigms of carbon fluxes and change in pH because of primary productivity. As a result, the article introduces the notion that HABs formation are not related to agricultural land use and water residence time and generate a great risk for the management of surface waterbodies. This reply also emphasizes the need for good practices of open data management, especially for public databases in view of external reproducibility.

Experimental evidence on the effects of temperature and salinity in morphological traits of the Microcystis aeruginosa complex.

Microcystis aeruginosa complex (MAC) encompasses noxious colonial bloom forming cyanobacteria. MAC representatives bloom in eutrophic freshwater and brackish ecosystems with stagnant water, were temperature and salinity are the main variables modulating their distribution, biomass and toxicity. Cell abundance and biovolume of MAC colonies define regulatory standards for public health. These variables depend upon colony size that in turn changes with environmental conditions. Here, we conducted two series of experiments to evaluate the response of MAC colonies morphological traits (length, volume, mucilage and number of cells) to temperature and salinity. In two series of experiments in the laboratory, we exposed natural MAC communities to three different temperatures (10, 21 and 30 °C) and four salinity levels (0, 5, 10 and 25 ppt) typically found in estuaries. We found that average colony length, volume and mucilage thickness did not change with temperature, but the cell-free space inside the colonies was smaller at the highest evaluated temperature (30 °C). Salinity fostered an increase in colony length, volume and mucilage thickness, while cell-free space diminished, resulting in higher cell density. The number of cells per colony was significantly related to colony size (length and volume) and both, temperature and salinity, affected the parameters of the relationships. Based on present results we propose statistical models to predict cell number per colony based on length and volume and accounting for the effect of salinity and temperature on these traits. This is applicable to ecological studies and to the monitoring of estuarine aquatic environments, by means of a fast and more accurate estimation of cell numbers to define MAC toxic populations early warning systems. A protocol is suggested for its application while the analysis of the interaction of temperature and salinity, as well as the variability in natural environments are objectives for future researches.

Improved biovolume estimation of Microcystis aeruginosa colonies: A statistical approach.

The Microcystis aeruginosa complex (MAC) clusters many of the most common freshwater and brackish bloom-forming cyanobacteria. In monitoring protocols, biovolume estimation is a common approach to determine MAC colonies biomass and useful for prediction purposes. Biovolume (µm3 mL-1) is calculated multiplying organism abundance (orgL-1) by colonial volume (µm3org-1). Colonial volume is estimated based on geometric shapes and requires accurate measurements of dimensions using optical microscopy. A trade-off between easy-to-measure but low-accuracy simple shapes (e.g. sphere) and time costly but high-accuracy complex shapes (e.g. ellipsoid) volume estimation is posed. Overestimations effects in ecological studies and management decisions associated to harmful blooms are significant due to the large sizes of MAC colonies. In this work, we aimed to increase the precision of MAC biovolume estimations by developing a statistical model based on two easy-to-measure dimensions. We analyzed field data from a wide environmental gradient (800 km) spanning freshwater to estuarine and seawater. We measured length, width and depth from ca. 5700 colonies under an inverted microscope and estimated colonial volume using three different recommended geometrical shapes (sphere, prolate spheroid and ellipsoid). Because of the non-spherical shape of MAC the ellipsoid resulted in the most accurate approximation, whereas the sphere overestimated colonial volume (3-80) especially for large colonies (MLD higher than 300 µm). Ellipsoid requires measuring three dimensions and is time-consuming. Therefore, we constructed different statistical models to predict organisms depth based on length and width. Splitting the data into training (2/3) and test (1/3) sets, all models resulted in low training (1.41-1.44%) and testing average error (1.3-2.0%). The models were also evaluated using three other independent datasets. The multiple linear model was finally selected to calculate MAC volume as an ellipsoid based on length and width. This work contributes to achieve a better estimation of MAC volume applicable to monitoring programs as well as to ecological research.

Using trait-based approaches to study phytoplankton seasonal succession in a subtropical reservoir in arid central western Argentina.

The application of trait-based approaches has become a widely applied tool to analyse community assembly processes and dynamics in phytoplankton communities. Its advantages include summarizing information of many species without losing essentials of the main driving processes. Here, we used trait-based approaches to study phytoplankton temporal succession in a subtropical reservoir. We applied a combined approach including morphological traits (i.e. volume, surface) and functional clustering of species (morphology-based functional groups (MBFG) and Reynolds' groups) and related the clustering of species with the environment. We found that this reservoir is characterized by a low richness and a bimodal distribution of phytoplankton biomass. Taxonomic and functional classifications were coincident, and the dominant species and groups biomasses were explained by the same group of variables. For instance, group X2, MBFG V and Carteria sp. biomasses were explained by: pH, Secchi disk depth, N-NH4; while group B, MBFG VI and Cyclotella ocellata biomasses were explained by stability of the water column, incident solar radiation, Secchi disk depth and N-NH4. From our results, we state that functional and taxonomic classifications are complementary rather than opposed approaches, and their specific uses depend exclusively on the aim of the study and the characteristics of the environment under evaluation. Our work is the first description of phytoplankton dynamics in a reservoir in the arid central western Argentina (Cuyo region).

Competition drives clumpy species coexistence in estuarine phytoplankton.

Understanding the mechanisms that maintain biodiversity is a fundamental problem in ecology. Competition is thought to reduce diversity, but hundreds of microbial aquatic primary producers species coexist and compete for a few essential resources (e.g., nutrients and light). Here, we show that resource competition is a plausible mechanism for explaining clumpy distribution on individual species volume (a proxy for the niche) of estuarine phytoplankton communities ranging from North America to South America and Europe, supporting the Emergent Neutrality hypothesis. Furthermore, such a clumpy distribution was also observed throughout the Holocene in diatoms from a sediment core. A Lotka-Volterra competition model predicted position in the niche axis and functional affiliation of dominant species within and among clumps. Results support the coexistence of functionally equivalent species in ecosystems and indicate that resource competition may be a key process to shape the size structure of estuarine phytoplankton, which in turn drives ecosystem functioning.

Implementation and evaluation of an undergraduate emergency medicine curriculum.

OBJECTIVE: To describe the implementation and evaluation of an undergraduate course in the first Australian academic emergency medicine unit. METHODS: A descriptive study of a course involving fifth year medical students at the University of Western Australia was undertaken. Teaching included self-directed case problem solving, small group tutorials, practical-skills teaching, clinical attachments and information handouts. Evaluation involved questionnaire scores and written feedback regarding life-support skills, tutorial teaching, course materials, clinical attachments and the course in general. Some groups of students underwent pre-course and post-course examinations. RESULTS: Subjective and objective testing showed that student knowledge significantly improved. Feedback was especially positive toward clinical attachments in emergency departments, practical skills tutorials and the case-based learning method. Students requested longer attachments to emergency departments, and more practical, case-based, interactive and bedside teaching. Problems encountered included inadequate time for teaching, vagueness about student roles and objectives, and dealing with death for the first time without adequate preparation. CONCLUSION: Undergraduate emergency medicine education should become an essential part of Australian and international undergraduate medical education. Emergency medicine is enjoyable and eminently suitable for problem-based, interactive and integrated teaching and improves confidence, clinical experience in emergencies, practical skills and teamwork. Improvements include more problem-based teaching, more practical skills sessions and better definition of student roles. These are general principles that can be applied to other undergraduate courses and to designers of other emergency medicine courses, both undergraduate and postgraduate.

Synthesis and biological properties of side-chain-modified bleomycins.

New side-chain-modified bleomycins (BLMs) 3a-k have been synthesized by the reaction of demethyl BLM A2 with alpha-bromoacetamides (2a-k). The structures of these BLM analogues have been established by comparison of their NMR spectra with the corresponding spectra of model thiazole derivatives. Mass spectra (FAB) of the modified BLMs are not informative, since the fragmentation patterns exhibit a loss of the modified chain moiety, presumably in the matrix. The purity of the compounds is attested by TLC and HPLC analyses. Biological evaluation of 3a-k in in vitro (survival of B16 melanoma cells) shows that the compounds are almost as effective as bleomycin. Examination of the effects of 3c, 3e, and 3f on lungs of male mice indicates that the analogues do not exhibit lower pulmonary toxicity than bleomycin.

Complete assignment of the 500 MHz 1H-NMR spectra of bleomycin A2 in H2O and D2O solution by means of two-dimensional NMR spectroscopy.

1H-NMR spectra of bleomycin A2 recorded at 500 MHz in D2O and H2O at 24 degrees C and 3 degrees C were investigated. Resonances of the individual spin systems were identified by using two-dimensional correlation spectroscopy (COSY), two-dimensional spin echo correlated spectroscopy (SECSY) and by the application of two-dimensional Nuclear Overhauser Effect spectroscopy (NOESY). Employment of these techniques allowed the assignment of 113 exchangeable and 59 non-exchangeable protons in the 1H NMR spectrum of bleomycin A2. By means of 2D NOE spectroscopy also interresidual connectivities could be observed. Comparison of the NOESY spectra at 3 degrees C and 24 degrees C suggest that at low temperatures the central party of the bleomycin A2 molecule tends to adopt an extended conformation.