Microplastics impacts in seven flagellate microalgae: Role of size and cell wall.

The toxicity of microplastic particles (MPs) on aquatic environments has been widely reported; however, their effects on protists are still contradictory. For example, it is unclear if cell size and cell wall have a role in shaping the response of flagellates to MPs. In this study, seven marine flagellated microalgae (six Dinoflagellates and one Raphidophyceae) were incubated with 10 mg L-1 MPs (polystyrene plastic micro-spheres, 1 µm diameter) to address the above question by measuring different response variables, i.e., growth, optimal photochemical efficiency (Fv/Fm), chlorophyll-a (Chl-a) content, superoxide dismutase (SOD) activity, and cell morphology. The effect of MPs on growth and Fv/Fm showed species-specificity effects. Maximum and minimum MPs-induced inhibitions were detected in Karenia mikimotoi (76.43%) and Akashiwo sanguinea (10.16%), respectively, while the rest of the species showed intermediate responses. The presence of MPs was associated with an average reduction of Chl-a content in most cases and with a higher superoxide dismutase activity in all cases. Seven species were classified into two groups by the variation of Chl-a under MPs treatment. One group (Prorocentrum minimum and Karenia mikimotoi) showed increased Chl-a, while the other (P. donghaiense, P. micans, Alexandrium tamarense, Akashiwo sanguinea, Heterosigma akashiwo) showed decreased Chl-a content. The MPs-induced growth inhibition was negatively correlated with cell size in the latter group. SEM images further indicated that MPs-induced malformation in the smaller cells (e.g., P. donghaiense and K. mikimotoi) was more severe than the bigger cells (e.g., A. sanguinea and P. micans), probably due to a relatively higher ratio of the cell surface to cell volume in the former. These results implicate that the effect of MPs on marine flagellated microalgae was related to the cell size among most species but not cell wall. Thus plastic pollution may have size-dependent effects on phytoplankton in future scenarios.

The toxic dinoflagellate Alexandrium catenella adversely affects early life stages of tehuelche scallop.

The effects of the toxic dinoflagellate Alexandrium catenella were investigated on growth, survival, and histopathology in larvae and spat of the Tehuelche scallop Aequipecten tehuelchus from Patagonia, Argentina. The study consisted of laboratory incubations of scallop larvae/spat with A. catenella, using environmentally realistic abundances of the dinoflagellate. Survival, growth, and histopathological effects were documented for scallop larvae/spat before, during, and after 7-day-long exposure to A. catenella. The scallops were grouped in flasks containing 0 (control), 20, 200, and 2000 cells mL-1 of A. catenella. The presence of A. catenella induced reduced larvae survival after 24 h, whereas a clear effect was observed after 3 days (survival of control larvae 95%, 72, and 79% for 20 and 200 cells mL-1, respectively, and 43% for 2000 cells mL-1). The growth rates of the control larvae and those exposed to 20 mL-1 cells were significantly different from zero. Histopathological effects (melanization, loss of connective tissue, necrosis, and inflammatory responses) were observed in spat exposed to A. catenella. These effects were more pronounced at the highest dinoflagellate concentration. Blooms of A. catenella frequently coincide with the reproductive season of A. tehuelchus, thus there is a need to further study the relationship between harmful algal blooms and the effect on scallops' natural populations in the region.

Effects of ocean acidification and phosphate limitation on physiology and toxicity of the dinoflagellate Karenia mikimotoi.

This work demonstrated a 10-day batch culture experiment to test the physiology and toxicity of harmful dinoflagellate Karenia mikimotoi in response to ocean acidification (OA) under two different phosphate concentrations. Cells were previously acclimated in OA (pH = 7.8 and CO2 = 1100 µatm) condition for about three months before testing the responses of K. mikimotoi cells to a two-factorial combinations experimentation. This work measured the variation in physiological parameters (growth, rETR) and toxicity (hemolytic activity and its toxicity to zebrafish embryos) in four treatments, representing two factorial combinations of CO2 (450 and 1100 µatm) and phosphate concentration (37.75 and 4.67 umol l-1). Results: OA stimulated the faster growth, and the highest rETRmax in high phosphate (HP) treatment, low phosphate (LP) and a combination of high CO2 and low phosphate (HC*LP) inhibited the growth and Ek in comparison to low CO2*high phosphate (LCHP) treatment. The embryotoxicity of K. mikimotoi cells enhanced in all high CO2 (HC) conditions irrespective of phosphate concentration, but the EC50 of hemolytic activity increased in all high CO2 (HC) and low phosphate (LP) treatments in comparison of LCHP. Ocean acidification (high CO2 and lower pH) was probably the main factor that affected the rETRmax, hemolytic activity and embryotoxicity, but low phosphate was the main factor that affected the growth, α, and Ek. There were significant interactive effects of OA and low phosphate (LP) on growth, rETRmax, and hemolytic activity, but there were no significant effects on α, Ek, and embryotoxicity. If these results are extrapolated to the aquatic environment, it can be hypothesized that the K. mikimotoi cells were impacted significantly by future changing ocean (e.g., ocean acidification and nutrient stoichiometry).

Effects of ocean acidification and solar ultraviolet radiation on physiology and toxicity of dinoflagellate Karenia mikimotoi.

A batch culture experiment was conducted to study the interactive effects of ocean acidification (OA) and solar ultraviolet radiation (UVR, 280-400 nm) on the harmful dinoflagellate Karenia mikimotoi. Cells were incubated in 7-days trials under four treatments. Physiological (growth, pigments, UVabc) and toxicity (hemolytic activity and its toxicity to zebrafish embryos) response variables were measured in four treatments, representing two factorial combinations of CO2 (400 and 1000 µatm) and solar irradiance (with or without UVR). Toxic species K. mikimotoi showed sustained growth in all treatments, and there was not statistically significant difference among four treatments. Cell pigment content decreased, but UVabc and hemolytic activity increased in all HC treatments and PAB conditions. The toxicity to zebrafish embryos of K. mikimotoi was not significantly different among four treatments. All HC and UVR conditions and the combinations of HC*UVR (HC-PAB) positively affected the UVabc, hemolytic activity in comparison to the LC*P (LC-P) treatment, and negatively affected the pigments. Ocean acidification (OA) was probably the main factor that affected the chlorophyll-a (Chl-a) and UVabc, but UVR was the main factor that affected the carotenoid (Caro) and hemolytic activity. There were no significant interactive effects of OA*UVR on growth, toxicity to zebrafish embryos. If these results are extrapolated to the natural environment, it can be hypothesized that this strain (DP-C32) of K. mikimotoi cells have the efficient mechanisms to endure the combination of ocean acidification and solar UVR. It is assumed that this toxic strain could form harmful bloom and enlarge the threatening to coastal communities, marine animals, even human health under future conditions.

Transcriptome sequencing of a toxic dinoflagellate, Karenia mikimotoi subjected to stress from solar ultraviolet radiation.

Solar ultraviolet radiation (UVR) is a stress factor in aquatic environments and may act directly or indirectly on orgnisms in the upper layers of the water column. However, UVR effects are usually species-specific and difficult to extrapolate. Here we use the HAB-forming, toxic dinoflagellate Karenia mikimotoi (which was found to be relatively resistant in previous studies) to investigate its transcriptional responses to a one-week UVR exposure. For this, batch cultures of K. mikimotoi were grown with and without UVR, and their transcriptomes (generated via RNAseq technology) were compared. RNA-seq generated 45.31 million reads, which were further assembled to 202600 unigenes (>300bp). Among these, ca. 61% were annotated with NCBI, NR, GO, KOG, PFAM, Swiss-Prot, and KEGG database. Transcriptomic analysis revealed 722 differentially expressed unigenes (DEGs, defined as being within a |log2 fold change| ≥ 2 and padj < 0.05) responding to solar UVR, which were only 0.36% of all unigenes. 716 unigenes were down-regulated, and only 6 unigenes were up-regulated in the UVR compared to non-UVR treatment. KEGG pathway further analysis revealed DEGs were involved in the different pathway; genes involved in the ribosome, endocytosis and steroid biosynthesis pathways were highly down-regulated, but this was not the case for those involved in the energy metabolisms (including photosynthesis, oxidative phosphorylation) which may contribute to the sustainable growth observed in UVR treatment. The up-regulated expression of both zinc-finger proteins (ZFPs) and ribosomal protein L11 (RPL11) may be one of the acclimated mechanisms against UVR. In addition, this work identified down-regulated genes involved in fatty acid degradation and the hydrophobic branched chain amino acids (e.g., Valine, leucine, and isoleucine), which act as structural components of cell membranes modulating lipid homeostasis or turnover. In conclusion, the present study suggests that the toxic dinoflagellate K. mikimotoi has limited transcriptomic regulation but confirms that it appears as a tolerant species in response to solar UVR. These findings expand current knowledge of gene expression in HAB-forming species in response to natural environment factors such as solar radiation.

Motility of Daphnia spinulata as affected by solar radiation throughout an annual cycle in mid-latitudes of Patagonia.

During an annual cycle, Daphnia spinulata collected from a plankton community of Patagonia was exposed outdoors to assess the impact of recently received solar radiation on motility (i.e. swimming speed and gravitaxis). Individual values of these parameters were obtained by video recordings and image analysis at different time intervals during the day. Initial swimming speed varied throughout the year, and changes in speed during exposure were not significantly affected by any waveband used in our experimental design (i.e. PAB, 280-700 nm; PA, 320-700 nm; and P, 400-700 nm). Overall, most of the individuals swam downwards, regardless of the radiation treatment imposed to the samples. We found that multifactor interactions (i.e. not a single parameter explained more than 40% of the observed variability) explained most of our observations on motility parameters. These factors include not only solar radiation, but other physical (underwater radiation field and wind intensity) and biological parameters (food availability, presence of predators and congeners). Our findings indicate that the plankton dynamics in the study site is likely to be governed by a sum of factors which must be taken into account when considering solar radiation effects on aquatic ecosystems.

Vertical migration and motility responses in three marine phytoplankton species exposed to solar radiation.

Diurnal vertical migration in the water column and the impact of solar radiation on motility were investigated in three marine phytoplankton species: Tetraselmis suecica, Dunaliella salina and Gymnodinium chlorophorum. Cells were exposed to solar radiation either in ultraviolet radiation (UVR, 280-400 nm) transparent Plexiglas tubes (45 cm length, 10 cm diameter) or in quartz tubes under three radiation treatments: PAB (280-700 nm), PA (320-700 nm) and P (400-700 nm). The three species displayed different behavior after exposure to solar radiation. Tetraselmis suecica was insensitive to UVR and under high solar radiation levels, cells accumulated preferentially near the surface. Exposure experiments did not indicate any significant changes in swimming speed nor in the percentage of motile cells after 5 h of exposure. On the other hand, D. salina was sensitive to UV-B displaying a significant decrease in swimming speed and percentage of motile cells after 2-3 h of exposure. Moreover, D. salina cells migrated deep in the water column when irradiance was high. The response of G. chlorophorum was in between that of the other two species tested, with a slight (but significant) decrease in swimming speed and percentage of motile cells in all radiation treatments after 5 h of exposure. While G. chlorophorum cells were more or less homogenously distributed in the water column, a slight (but significant) avoidance response to high radiation was observed at local noon, with cells migrating deep in the water column. Our data clearly indicate that these sub-lethal effects of solar radiation are species-specific and they might have important implications for the aquatic ecosystem.

Impact of solar ultraviolet radiation on marine phytoplankton of Patagonia, Argentina.

Patagonia area is located in close proximity to the Antarctic ozone "hole" and thus receives enhanced ultraviolet B (UV-B) radiation (280-315 nm) in addition to the normal levels of ultraviolet A (UV-A; 315-400 nm) and photosynthetically available radiation (PAR; 400-700 nm). In marine ecosystems of Patagonia, normal ultraviolet radiation (UVR) levels affect phytoplankton assemblages during the three phases of the annual succession: (1) prebloom season (late summer-fall), (2) bloom season (winter-early spring) and (3) postbloom season (late spring-summer). Small-size cells characterize the pre- and postbloom communities, which have a relatively high photosynthetic inhibition because of high UVR levels during those seasons. During the bloom, characterized by microplankton diatoms, photosynthetic inhibition is low because of the low UVR levels reaching the earth's surface during winter; this community, however, is more sensitive to UV-B when inhibition is normalized by irradiance (i.e. biological weighting functions). In situ studies have shown that UVR significantly affects not only photosynthesis but also the DNA molecule, but these negative effects are rapidly reduced in the water column because of the differential attenuation of solar radiation. UVR also affects photosynthesis versus irradiance (P vs E) parameters of some natural phytoplankton assemblages (i.e. during the pre- but not during the postbloom season). However, there is a significant temporal variability of P vs E parameters, which are influenced by the nutrient status of cells and taxonomic composition; taxonomic composition is in turn associated with the stratification conditions (e.g. wind speed and duration). In Patagonia, wind speed is one of the most important variables that conditions the development of the winter bloom by regulating the depth of the upper mixed layer (UML) and hence the mean irradiance received by cells. Studies on the interactive effects of UVR and mixing show that responses of phytoplankton vary according to the taxonomic composition and cell structure of assemblages; therefore cells use UVR if >90% of the euphotic zone is being mixed. In fact, cell size plays a very important role when estimating the impact of UVR on phytoplankton, with large cells being more sensitive when determining photosynthesis inhibition, whereas small cells are more sensitive to DNA damage. Finally, in long-term experiments, it was determined that UVR can shape the diatom community structure in some assemblages of coastal waters, but it is virtually unknown how these changes affect the trophodynamics of marine systems. Future studies should consider the combined effects of UVR on both phytoplankton and grazers to establish potential changes in biodiversity of the area.

DNA damage and primordial follicle activation after in vitro culture of sheep ovarian cortex in Morus nigra leaf extract / Dano ao DNA e ativação do folículo primordial após a cultivo in vitro do córtex ovariano de ovelha em extrato de folha de Morus nigra

This study evaluated the effect of Morus nigra leaf extract, with or without supplementation, on morphology, activation and DNA damage of preantral follicles cultured within sheep ovarian tissue. Ovaries were collected and divided into fragments, being one fixed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) analysis (fresh control). The remaining fragments were cultured for 7 days in alpha minimum essential media (α-MEM) supplemented with bovine serum albumin (BSA), insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (α-MEM+; control medium) or into medium composed of M. nigra extract without supplements (0.1; 0.2 or 0.4mg/mL) or supplemented with the same substances described above for α-MEM+ (MN 0.1+; 0.2+ or 0.4+mg/mL). Then, tissues were destined to histological and TUNEL analysis. The α-MEM+ treatment had more morphologically normal follicles than all M. nigra extract treatments. However, α-MEM+ treatment also showed signs of atresia because the percentage of TUNEL positive cells was similar in α-MEM+ and in 0.1mg/mL M. nigra without and with supplements. Moreover, a reduction in the primordial follicles and an increase in the growing ones were observed in all treatments, except 0.2mg/mL M. nigra. In conclusion, the follicles cultured at 0.1mg/mL M. nigra extract were in good condition and able to continue their development, as demonstrated by the same rates of DNA damage and follicular activation as the control medium.(AU)

Este estudo avaliou o efeito do extrato das folhas de Morus nigra, com ou sem suplementos, sobre a morfologia, a ativação e o dano ao DNA de folículos pré-antrais cultivados inclusos em tecido ovariano. Os ovários foram coletados e divididos em fragmentos, sendo um fixado para análise histológica e ensaio de marcação de terminações dUTP mediada por desoxinucleotidil transferase terminal (TUNEL) (controle fresco). Os fragmentos restantes foram cultivados durante 7 dias em meio essencial mínimo alfa (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, transferrina, selênio, glutamina, hipoxantina e ácido ascorbico (α-MEM+; meio controle) ou em meio composto de extrato de M. nigra sem suplementos (0,1; 0,2 or 0,4mg/mL) ou suplementado com as mesmas substâncias descritas para α-MEM+ (MN 0,1+; 0,2+ or 0,4+mg/mL). Então, os tecidos foram destinados à análise histológica e TUNEL. O tratamento do α-MEM+ apresentou mais folículos morfologicamente normais que todos os tratamentos do extrato de M. nigra. No entanto, o tratamento com α-MEM+ também mostrou sinais de atresia, pois a porcentagem de células TUNEL positivas foi semelhante em α-MEM+ e em 0,1mg/mL M. nigra sem e com suplementos. Além disso, observou-se uma redução nos folículos primordiais e um aumento nos folículos em crescimento em todos os tratamentos, exceto 0,2mg/mL M. nigra. Em conclusão, os folículos cultivados com 0,1mg/mL de extrato de M. nigra estavam em boas condições e aptos a continuar seu desenvolvimento, como demonstrado pelas taxas de dano ao DNA e de ativação folicular semelhantes ao meio controle.(AU)

Photorepair activity and protective compounds in two freshwater zooplankton species (Daphnia menucoensis and Metacyclops mendocinus) from Patagonia, Argentina.

The impact of ultraviolet-B radiation (UV-B, 280-315 nm) on the cladoceran Daphnia menucoensis Paggi and the copepod Metacyclops mendocinus (Wierzejski) was determined in experiments designed to evaluate the effectiveness of the photorepair mechanism and the role of UV-absorbing compounds. In both species UV-B caused significant mortality at doses of approximately 40 kJ m(- 2) or higher. At lower UV-B doses, however, no significant mortality was detected in M. mendocinus; moreover, this species seems to have a threshold below which no UV-B induced mortality is determined. D. menucoensis, on the other hand, was very sensitive to UV-B, and significant mortality of 15% (p < 0.05) was observed when doses were as low as 10 kJ m(-2). Both species showed high efficiency for photorepairing UV-B-induced damage to the DNA molecule, with a significant decrease of mortality when the species were exposed to visible radiation, PAR (55 W m(-2)), in addition to UV-B. The higher resistance of M. mendocinus to UV-B as compared to that of D. menucoensis might be also related to the presence of mycosporine-like amino acids, MAAs (i.e., shinorine and porphyra-334), and carotenoids, which would add an adaptive advantage to the copepod.