Updating embryonic ontogenesis in Araucaria angustifolia: from Burlingame (1915) to the present.

This study addresses gaps in our understanding of pre-fertilization and archegonia development and reinterprets embryonic ontogenesis from Burlingame (Bot Gaz 59:1-39, 1915) to the present based on timescale and structural features allowing us to determine functionally and developmentally accurate terminology for all these stages in A. angustifolia. Different from previous reports, only after pollination, pre-fertilization tissue development occurs (0-13 months after pollination (MAP)) and gives rise to a mature megagametophyte. During all this period, pollen is in a dormant state at the microphyla, and pollen tube germination in nucellus tissue is only observed at the stage of archegonia formation (13 MAP) and not at the free nuclei stage as reported before. For the first time, 14 months after pollination, a fertilization window was indicated, and at 15 MAP, the polyzygotic polyembryony from different archegonia was also seen. After that, subordinated proembryo regression occurs and at least three embryonic developmental stages of dominant embryo were characterized: proembryogenic, early embryogenic, and late embryogenic (15-23 MAP). Along these stages, histochemical and ultrastructural analyses suggest the occurrence of cell death in suspensor and in cap cells of dominant embryo that was not previously reported. The differentiation of meristems, procambium, pith, and cortex tissues in late embryogenic stage was detailed. The morphohistological characterization of pre-fertilization and embryonic stages, together with the timescale of megastrobili development, warranted a referential map of female reproductive structure in this species.

Effects of phenanthrene on early development of the Pacific oyster Crassostrea gigas (Thunberg, 1789).

Phenanthnere (PHE) is a polycyclic aromatic hydrocarbon continuously discarded in the marine environment and bioavailable to many aquatic species. Although studies about PHE toxicity have been documented for adult oysters, the effects on early developmental stages are poorly characterized in bivalves. In this study, the effects of PHE (0.02 and 2.0µg.L-1) were evaluated on the embryogenesis and larval development of Crassostrea gigas. Toxicity bioassays, growth and deformities assessment, analysis of shell calcium abundance and transcript levels of genes related to xenobiotic biotransformation (CYP2AU2, CYP30C1), immune system (Cg-Tal) and tissue growth and shell formation (Ferritin, Insulin-like, Cg-Try, Calmodulin and Nacrein) were assayed in D-shape larvae after 24h of PHE exposure. At the highest concentration (2.0µg.L-1), PHE decreased the frequency of normal development (19.7±2.9%) and shell size (53.5±2.8mm). Developmental deformities were mostly related to abnormal mantle and shell formation. Lower calcium levels in oyster shells exposed to PHE 2.0µg.L-1 were observed, suggesting effects on shell structure. At this same PHE concentration, CYP30C1, Cg-Tal, Cg-Tyr, Calmodulin were upregulated and CYP2AU2, Ferritin, Nacrein, and Insulin-Like were downregulated compared to control larvae. At the lowest PHE concentration (0.02µg.L-1), it was observed a minor decrease in normal larval development (89,6±6%) and the remaining parameters were not affected. This is the first study to provide evidences that exposure to PHE can affect early oyster development at the molecular and morphological levels, possibly threatening this bivalve species.

The brown seaweed Sargassum cymosum: changes in metabolism and cellular organization after long-term exposure to cadmium.

Sargassum cymosum was exposed to cadmium (Cd) to determine any physiological and ultrastructural effects. To accomplish this, S. cymosum samples were cultivated under photosynthetic active radiation (PAR) and Cd (0, 0.1, 0.2, 0.4 and 0.8 mg L-1) during 7 and 14 days in laboratory-controlled conditions (0 mg L-1 Cd at both exposure times as control). Seaweeds had high retention capacity (over 90 %) for both exposure times. Growth rates showed significant increases by 14 days, especially for 0.1 and 0.4 mg L-1 Cd. Photosynthetic parameters were unaffected by Cd treatments. Chlorophyll contents were present in higher concentrations for all Cd treatments compared to respective control. Carotenoid profile showed significant differences in total composition and proportion of fucoxanthin and ß-carotene, and no lutein was detected at 14 days. Phenolic and flavonoid compounds showed major accumulation at 14 days. Transmission electron microscopy (TEM) analyses presented major alterations in Cd-treated samples, when compared with respective control, in particular disorganization of cell wall fibrils. When compared to respective control samples, multivariate analyses showed disparate and complex interactions among metabolites in Cd-exposed seaweeds, giving evidence of physiological defence response. Thus, it can be concluded that Cd is a stressor for S. cymosum, resulting in physiological and structural alterations related to defence mechanisms against oxidative stress and toxicological effects resulting from long-term metal exposure. However, in the present paper, some observed changes also appear to result from acclimation mechanisms under lower concentration of Cd relative to the tolerance of S. cymosum to experimental conditions.

The influence of salinity on growth, morphology, leaf ultrastructure, and cell viability of the seagrass Halodule wrightii Ascherson.

Halodule wrightii is an ecologically important seagrass; however, little is known about the adaptation of this species in the context of environmental change, particularly changes arising from alterations in salinity of coastal ecosystems. This study aimed to determine the effects of different salinities on growth, morphology, leaf ultrastructure, and cell viability of H. wrightii. To accomplish this, plants were cultivated for 21 days in salinities of 25, 35, and 45. More hydropotens were observed in samples exposed to salinity of 45 with increased invagination of the plasma membrane and cell wall. These invaginations were also observed in other epidermal cells of the leaf blade. In particular, a significant retraction of plasma membrane was seen in samples exposed to salinity of 45, with possible deposition of compounds between the membrane and cell wall. Osmotic stress in samples exposed to salinity of 45 affected the chloroplasts through an increase in plastoglobules and thylakoids by granum in the epidermal chloroplasts of the leaf and decrease in the number of chloroplasts. Overall, this study showed that H. wrightii can survive within salinities that range between 25 and 45 without changing growth rate. However, the plant did have higher cell viability at salinity of 35. Salt stress in mesocosms, at both salinity of 25 and 45, decreased cell viability in this species. H . wrightii had greater changes in salinity of 45; this showed that the species is more tolerant of salinities below this value.

Synthesis, characterization and toxicological evaluation of Cr2O3 nanoparticles using Daphnia magna and Aliivibrio fischeri.

Chromium III oxide (Cr2O3) nanoparticles (NPs) are used in pigments for ceramics, dyes, paints and cosmetics. However, few studies addressing the toxic potential of these NPs have been reported in the literature. Thus, this research aimed to evaluate the acute and chronic effects of Cr2O3 NPs through acute toxicity tests with Daphnia magna and Aliivibrio fischeri and chronic toxicity tests with Daphnia magna. Cr2O3 NPs were synthesized by the sol-gel method and characterized through TEM, X-Ray diffraction (XRD), zeta potential (ZP) and surface area analysis. In the acute toxicity tests the EC(50,48h) value obtained with D. magna was 6.79 mg L(-1) and for A. fischeri the EC(50,15min) value was 16.10 mg L(-1) and the EC(50,30min) value was 12.91 mg L(-1). Regarding the chronic toxicity tests with D. magna, effects on longevity (OEC=1.00 mg L(-1)), reproduction (OEC=1.00 mg L(-1)) and growth (OEC=0.50 mg L(-1)) were observed. On the SEM and TEM images, ultrastructural alterations in the organelles of exposed organisms were also observed. Thus, toxicological studies with NPs are of great importance in order to reduce the risk of environmental contamination.

Seasonal biochemical and photophysiological responses in the intertidal macroalga Cystoseira tamariscifolia (Ochrophyta).

Seasonal changes in the biochemistry and photophysiology of the brown macroalga Cystoseira tamariscifolia was analyzed in southern Spain. Total carbon and nitrogen contents, phenolic compounds, antioxidant and photosynthetic activities were seasonally determined over two years. Carbon, nitrogen and photoprotective phenolic contents were higher in winter and spring than in summer and autumn. Antioxidant levels were highest in spring and we found a positive correlation between phenolic content and antioxidant activity (EC50). Photosynthetic capacity (ETRmax) and photosynthetic efficiency (αETR) were also highest in spring, and there was a positive correlation between ETRmax and the amount of phenols present. Increased irradiance in spring enhanced algal productivity, antioxidant activity and the production of photoprotective compounds but in summer nutrient depletion due to thermal stratification of coastal waters reduced photosynthetic activity and the photoprotective capacity of C. tamariscifolia. Electron microscopy showed that phenols occurred in the cytoplasm of cortical cells inside physodes. Spring would be the best period to harvest C. tamariscifolia to extract photoprotectors and antioxidants for potential commercial uses, although the environmental impacts would need to be carefully assessed.

Effects of copper and lead exposure on the ecophysiology of the brown seaweed Sargassum cymosum.

The effects of the heavy metals copper (Cu) and lead (Pb) on Sargassum cymosum were evaluated by determining uptake capacity, growth rates, photosynthetic efficiency, contents of photosynthetic pigments and phenolic compounds, 2,2-diphenyl-1-picrylhydrazyl radical-scavenging capacity, and morphological and cellular changes. S. cymosum was cultivated with Cu and Pb separately and combined at concentrations of 10, 25, and 50 µM for 7 days in laboratory-controlled conditions. Seaweeds under Cu treatment showed the highest biosorption capacity, and growth rates were significantly reduced compared to the control. The photosynthesis/irradiance curves showed alterations in kinetic patterns in the metal-treated samples. Specifically, Cu treatment alone inhibited electron transport rate (ETR) response, while Pb alone induced it. However, samples treated with both Cu and Pb (Cu + Pb) showed inhibition in ETR. The total amount of pigments increased relative to control. Light microscopy showed an increase in phenolic compounds, with physodes migrating towards cortical cells. Scanning electronic microscopy revealed alterations in the typical rough surface of thallus, when compared with control, especially for Pb treatments. Based on these results, it could be concluded that Cu and Pb are stress factors for S. cymosum, promoting alterations in seaweed metabolism and stimulating protective mechanisms against oxidative stress. However, the high bioaccumulation capacity of both heavy metals indicates a possible application for S. cymosum as a biosorbent agent for contaminated wastewater when metals are in low concentrations.

Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of Araucaria angustifolia (Bert.) O. Kuntze.

Somatic embryogenesis is a morphogenetic route useful for the study of embryonic development, as well as the large-scale propagation of endangered species, such as the Brazilian pine (Araucaria angustifolia). In the present study, we investigated the morphological and ultrastructural organization of A. angustifolia somatic embryo development by means of optical and electron microscopy. The proembryogenic stage was characterized by the proliferation of proembryogenic masses (PEMs), which are cellular aggregates composed of embryogenic cells (ECs) attached to suspensor-like cells (SCs). PEMs proliferate through three developmental stages, PEM I, II, and III, by changes in the number of ECs and SCs. PEM III-to-early somatic embryo (SE) transition was characterized by compact clusters of ECs growing out of PEM III, albeit still connected to it by SCs. Early SEs showed a dense globular embryonic mass (EM) and suspensor region (SR) connected by embryonic tube cells (TCs). By comparison, early somatic and zygotic embryos showed similar morphology. ECs are round with a large nucleus, nucleoli, and many cytoplasmic organelles. In contrast, TCs and SCs are elongated and vacuolated with cellular dismantling which is associated with programmed cell death of SCs. Abundant starch grains were observed in the TCs and SCs, while proteins were more abundant in the ECs. Based on the results of this study, a fate map of SE development in A. angustifolia is, for the first time, proposed. Additionally, this study shows the cell biology of SE development of this primitive gymnosperm which may be useful in evolutionary studies in this area.

Effects of UV-B radiation on germlings of the red macroalga Nemalion helminthoides (Rhodophyta).

Studies have clearly demonstrated the damaging effects of UV-B exposure on macroalgae, but few have reported the impact of UV-B on spore germination and development at juvenile stages. Therefore, this work aimed to analyze the effects of UV-B radiation on germlings of Nemalion helminthoides at the tetrasporophytic phase. To accomplish this, germlings of N. helminthoides were cultivated in the laboratory and separated into two groups. The control group was exposed onlyto photosynthetic radiation, while the treatment group was exposed to photosynthetic radiation + UV-B for 2 hours during a period of 12 days. Control germlings showed increasing cellular proliferation and accumulation of reserve substances, as well as intense ramification in the last observed stages between 9 days and 12 days of development. Moreover, the chloroplasts presented a typical globular pyrenoid, profusely traversed by thylakoid membranes. Treated germlings, by contrast, showed intracellular damage, such as cell wall thickness, loss of chloroplast organization, changes in mitochondrial cristae, and increasing atrophy of the Golgi bodies. Additionally, changes in developmental patterns were observed, including loss of polarity in the first divisions of carpospores and abnormal stem ramification. The quantification of autofluorescence data coincided with the ultrastructural changes observed in the chloroplasts of cells exposed to UV-B. It can be concluded that exposure to radiation changed the developmental pattern and morphology of the germlings of N. helminthoides.

Effects of eutrophic seawater and temperature on the physiology and morphology of Hypnea musciformis J. V. Lamouroux (Gigartinales, Rhodophyta).

As both food and source of a kappa-carrageenan, Hypnea musciformis represents a species of great economic interest. It also synthesizes substances with antiviral, anti-helminthic and anti-inflammatory potential and shows promise for use as a bioindicator of cadmium. In this study, we investigated the combined effects of seawater from three urbanized areas (area 1: natural runoff, NRA; area 2: urbanized runoff and sewage with treatment, RTA; area 3: urbanized runoff and untreated sewage, RUS) and three different temperatures (15, 25 and 30 °C) on the growth rate, photosynthetic efficiency, photosynthetic pigments and cell morphology of H. musciformis. After 4 days (96 h) of culture, the biomass of H. musciformis showed differences that fluctuated among the areas and temperature treatments. Specifically, the specimens cultivated in 35 °C had low values of ETRmax, α(ETR), ß(ETR), and Fv/Fm photosynthetic parameters, as well as changes in cell morphology, with reduction in photosynthetic pigments and drastic reduction in growth rates. When combined with the extreme temperatures, high concentrations of ammonium ion in seawater effluent caused an inhibition of photosynthetic activity, as well as significant variation in chlorophyll a and carotenoid contents. As observed by light microscopy, the synergism between different temperatures and pollutants found in eutrophic waters caused changes in cellular morphology with increased cell wall thickening and decreased floridean starch grains. H. musciformis also showed important changes in physiological response to each factor independently, as well as changes resulting from the synergistic interaction of these factors combined. Therefore, we can conclude that extreme temperature combined with the effect of eutrophic waters, especially RUS, caused distinct morphological and physiological changes in the red alga H. musciformis.

Effects of cadmium metal on young gametophytes of Gelidium floridanum: metabolic and morphological changes.

By evaluating carotenoid content, photosynthetic pigments and changes in cellular morphology, growth rates, and photosynthetic performance, this study aimed to determine the effect of cadmium (Cd) on the development of young gametophytes of Gelidium floridanum. Plants were exposed to 7.5 and 15 µM of Cd for 7 days. Control plants showed increased formation of new filamentous thallus, increased growth rates, presence of starch grains in the cortical and subcortical cells, protein content distributed regularly throughout the cell periphery, and intense autofluorescence of chloroplasts. On the other hand, plants treated with Cd at concentrations of 7.5 and 15 µM showed few formations of new thallus with totally depigmented regions, resulting in decreased growth rates. Plants exposed to 7.5 µM Cd demonstrated alterations in the cell wall and an increase in starch grains in the cortical and subcortical cells, while plants exposed to 15 µM Cd showed changes in medullary cells with no organized distribution of protein content. The autofluorescence and structure of chloroplasts decreased, forming a thin layer on the periphery of cells. Cadmium also affected plant metabolism, as visualized by a decrease in photosynthetic pigments, in particular, phycoerythrin and phycocyanin contents, and an increase in carotenoids. This result agrees with decreased photosynthetic performance and chronic photoinhibition observed after treatment with Cd, as measured by the decrease in electron transport rate. Based on these results, it was concluded that exposure to Cd affects cell metabolism and results in significant toxicity to young gametophytes of G. floridanum.

The effect of different concentrations of copper and lead on the morphology and physiology of Hypnea musciformis cultivated in vitro: a comparative analysis.

Copper and lead, as remnants of industrial activities and urban effluents, have heavily contaminated many aquatic environments. Therefore, this study aimed to determine their effects on the physiological, biochemical, and cell organization responses of Hypnea musciformis under laboratory conditions during a 7-day experimental period. To accomplish this, segments of H. musciformis were exposed to photosynthetic active radiation at 80 µmol photons m(-2) s(-1), Cu (0.05 and 0.1 mg kg(-1)), and Pb (3.5 and 7 mg kg(-1)). Various intracellular abnormalities resulted from exposure to Cu and Pb, including a decrease in phycobiliproteins. Moreover, carotenoid and flavonoid contents, as well as phenolic compounds, were decreased, an apparent reflection of chemical antioxidant defense against reactive oxygen species. Treatment with Cu and Pb also caused an increase in the number of floridean starch grains, probably as a defense against nutrient deprivation. Compared to plants treated with lead, those treated with copper showed higher metabolic and ultrastructural alterations. These results suggest that H. musciformis more readily internalizes copper through transcellular absorption. Finally, as a result of ultrastructural damage and metabolic changes observed in plants exposed to different concentrations of Cu and Pb, a significant reduction in growth rates was observed. Nevertheless, the results indicated different susceptibility of H. musciformis to different concentrations of Cu and Pb.

Histodifferentiation and ultrastructure of nodular cultures from seeds of Vriesea friburgensis Mez var. paludosa (L.B. Smith) L.B. Smith and leaf explants of Vriesea reitzii Leme & A. Costa (Bromeliaceae).

Micropropagation via induction, multiplication and development of nodular cultures (NCs) is an efficient regeneration system for Bromeliaceae, a family of endangered monocot plants with ornamental value. Therefore, the present work aimed to induce NCs from seeds and leaf explants of Vriesea in order to characterize the morphological and histochemical aspects of induction and formation of these cultures. Seeds of Vriesea friburgensis var. paludosa were sterilized and inoculated into liquid culture media supplemented with different concentrations and combinations of growth regulators. Leaf explants of Vriesea reitzii were inoculated into medium supplemented with 4 µM α-naphthalene acetic acid (NAA) and 2 µM 6-benzylaminopurine (BAP). The addition of NAA (4 µM) in the culture medium used for seeds led to an induction rate of 72% in NCs. First, the embryo began to germinate, and afterwards, nodular structures started to form. While NCs formed from seeds is associated with root and shoot meristems, the formation of NCs from leaf explants involves the intercalary meristem. Meristematic cells generate an appropriate response in the induction medium, producing NCs by the proliferation of small cells with meristematic characteristics and large vacuolated cells. These results provide a better understanding of morphogenetic responses in bromeliads and, hence, the opportunity to develop optimized micropropagation protocols. Abbreviations: BAP, 6-benzylaminopurine; 2-iP, N6 (2-isopentyl) adenine; CBB, Coomassie Brilliant Blue; CLSM, confocal laser scanning microscopy; MSB, MS basal medium; NAA, α-Naphthalene acetic acid; NCs, nodular cultures; PAS, Periodic Acid-Schiff; SEM, scanning electron microscopy; TDZ, N-phenyl-N'-1,2,3-thidiazol-5-ylurea; TB-O, Toluidine Blue O; TEM, Transmission electron microscopy.

Effects of ultraviolet radiation (UVA+UVB) and copper on the morphology, ultrastructural organization and physiological responses of the red alga Pterocladiella capillacea.

The effect of ultraviolet (UV) radiation and copper (Cu) on apical segments of Pterocladiella capillacea was examined under two different conditions of radiation, PAR (control) and PAR+UVA+UVB (PAR+UVAB), and three copper concentrations, ranging from 0 (control) to 0.62, 1.25 and 2.50 µm. Algae were exposed in vitro to photosynthetically active radiation (PAR) at 70 µmol photons m(-2)  s(-1) , PAR + UVB at 0.35 W m(-2) and PAR +UVA at 0.70 W m(-2) during a 12-h photocycle for 3 h each day for 7 days. The effects of radiation and copper on growth rates, content of photosynthetic pigments and photosynthetic performance were analyzed. In addition, samples were processed for light and transmission electron microscopy. The content of photosynthetic pigments decreased after exposure to radiation and Cu. Compared with PAR radiation and copper treatments modified the kinetics patterns of the photosynthesis/irradiance curve. The treatments also caused changes in the ultrastructure of cortical and subcortical cells, including increased cell wall thickness and accumulation of plastoglobuli, as well as changes in the organization of chloroplasts. The results indicate that the synergistic interaction between UV radiation and Cu in P. capillacea, led to the failure of protective mechanisms and causing more drastic changes and cellular imbalances.

Toxicological effects of copper oxide nanoparticles on the growth rate, photosynthetic pigment content, and cell morphology of the duckweed Landoltia punctata.

Recently, the application of copper oxide nanoparticles (CuO-NPs) has increased considerably, primarily in scientific and industrial fields. However, studies to assess their health risks and environmental impacts are scarce. Therefore, the present study aims to evaluate the toxicological effects of CuO-NPs on the duckweed species Landoltia punctata, which was used as a test organism. To accomplish this, duckweed was grown under standard procedures according to ISO DIS 20079 and exposed to three different concentrations of CuO-NPs (0.1, 1.0, and 10.0 g L(-1)), with one control group (without CuO-NPs). The toxicological effects were measured based on growth rate inhibition, changes in the plant's morphology, effects on ultrastructure, and alterations in photosynthetic pigments. The morphological and ultrastructural effects were evaluated by electronic, scanning and light microscopic analysis, and CuO-NPs were characterized using transmission electron microscopy (TEM), zeta potential, and superficial area methods of analysis. This analysis was performed to evaluate nanoparticle size and form in solution and sample stability. The results showed that CuO-NPs affected morphology more significantly than growth rate. L. punctata also showed the ability to remove copper ions. However, for this plant to be representative within the trophic chain, the biomagnification of effects must be assessed.

The effect of cadmium under different salinity conditions on the cellular architecture and metabolism in the red alga Pterocladiella capillacea (Rhodophyta, Gelidiales).

The in vitro effect of cadmium (Cd) on apical segments of Pterocladiella capillacea was examined. Over a period of 7 days, the segments were cultivated with the combination of different salinities (25, 35, and 45 practical salinity units) and Cd concentrations, ranging from 0.17 to 0.70 ppm. The effects of Cd on growth rates and content of photosynthetic pigments were analyzed. In addition, metabolic profiling was performed, and samples were processed for microscopy. Serious damage to physiological performance and ultrastructure was observed under different combinations of Cd concentrations and salinity values. Elementary infrared spectroscopy revealed toxic effects registered on growth rate, photosynthetic pigments, chloroplast, and mitochondria organization, as well as changes in lipids and carbohydrates. These alterations in physiology and ultrastructure were, however, coupled to activation of such defense mechanisms as cell wall thickness, reduction of photosynthetic harvesting complex, and flavonoid. In conclusion, P. capillacea is especially sensitive to Cd stress when intermediate concentrations of this pollutant are associated with low salinity values. Such conditions resulted in metabolic compromise, reduction of primary productivity, i.e., photosynthesis, and carbohydrate accumulation in the form of starch granules. Taken together, these findings improve our understanding of the potential impact of this metal in the natural environment.

Effects of ultraviolet radiation (UVA+UVB) on young gametophytes of Gelidium floridanum: growth rate, photosynthetic pigments, carotenoids, photosynthetic performance, and ultrastructure.

This study investigated the effects of radiation (PAR+UVA+UVB) on the development and growth rates (GRs) of young gametophytes of Gelidium floridanum. In addition, photosynthetic pigments were quantified, carotenoids identified, and photosynthetic performance assessed. Over a period of 3 days, young gametophytes were cultivated under laboratory conditions and exposed to photosynthetically active radiation (PAR) at 80 µmol photons m(-2) s(-1) and PAR+UVA (0.70 W m(-2))+UVB (0.35 W m(-2)) for 3 h per day. The samples were processed for light and electron microscopy to analyze the ultrastructure features, as well as carry out metabolic studies of GRs, quantify the content of photosynthetic pigments, identify carotenoids and assess photosynthetic performance. PAR+UVA+UVB promoted increase in cell wall thickness, accumulation of floridean starch grains in the cytoplasm and disruption of chloroplast internal organization. Algae exposed to PAR+UVA+UVB also showed a reduction in GR of 97%. Photosynthetic pigments, in particular, phycoerythrin and allophycocyanin contents, decreased significantly from UV radiation exposure. This result agrees with the decrease in photosynthetic performance observed after exposure to ultraviolet radiation, as measured by a decrease in the electron transport rate (ETR), where values of ETRmax declined approximately 44.71%. It can be concluded that radiation is a factor that affects the young gametophytes of G. floridanum at this stage of development.

Bioabsorption of cadmium, copper and lead by the red macroalga Gelidium floridanum: physiological responses and ultrastructure features.

Heavy metals, such as lead, copper, cadmium, zinc, and nickel, are among the most common pollutants found in both industrial and urban effluents. High concentrations of these metals cause severe toxic effects, especially to organisms living in the aquatic ecosystem. Cadmium (Cd), lead (Pb) and copper (Cu) are the heavy metals most frequently implicated as environmental contaminants, and they have been shown to affect development, growth, photosynthesis and respiration, and morphological cell organization in seaweeds. This paper aimed to evaluate the effects of 50µM and 100µM of Cd, Pb and Cu on growth rates, photosynthetic pigments, biochemical parameters and ultrastructure in Gelidium floridanum. To accomplish this, apical segments of G. floridanum were individually exposed to the respective heavy metals over a period of 7 days. Plants exposed to Cd, Cu and Pb showed discoloration of thallus pigmentation, chloroplast alteration, especially degeneration of thylakoids, and decrease in photosynthetic pigments, such as chlorophyll a and phycobiliproteins, in samples treated with Cd and Cu. Moreover, cell wall thickness and the volume of plastoglobuli increased. X-ray microanalysis detected Cd, Cu and Pb absorption in the cell wall. The results indicate that Cd, Pb and Cu negatively affect metabolic performance and cell ultrastructure in G. floridanum and that Cu was more toxic than either Pb or Cd.

Gills are an initial target of zinc oxide nanoparticles in oysters Crassostrea gigas, leading to mitochondrial disruption and oxidative stress.

The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnONP) was investigated in Pacific oysters Crassostrea gigas. The nanoscale of ZnONP, in vehicle or ultrapure water, was confirmed, presenting an average size ranging from 28 to 88 nm. In seawater, aggregation was detected by TEM and DLS analysis, with an increased average size ranging from 1 to 2 µm. Soluble or nanoparticulated zinc presented similar toxicity, displaying a LC50 (96 h) around 30 mg/L. High zinc dissociation from ZnONP, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONP toxicity. To investigate mechanisms of toxicity, oysters were treated with 4 mg/L ZnONP for 6, 24 or 48 h. ZnONP accumulated in gills (24 and 48 h) and digestive glands (48 h). Ultrastructural analysis of gills revealed electron-dense vesicles near the cell membrane and loss of mitochondrial cristae (6 h). Swollen mitochondria and a more conspicuous loss of mitochondrial cristae were observed after 24 h. Mitochondria with disrupted membranes and an increased number of cytosolic vesicles displaying electron-dense material were observed 48 h post exposure. Digestive gland showed similar changes, but these were delayed relative to gills. ZnONP exposure did not greatly affect thiol homeostasis (reduced and oxidized glutathione) or immunological parameters (phagocytosis, hemocyte viability and activation and total hemocyte count). At 24 h post exposure, decreased (-29%) glutathione reductase (GR) activity was observed in gills, but other biochemical responses were observed only after 48 h of exposure: lower GR activity (-28%) and levels of protein thiols (-21%), increased index of lipid peroxidation (+49%) and GPx activity (+26%). In accordance with ultrastructural changes and zinc load, digestive gland showed delayed biochemical responses. Except for a decreased GR activity (-47%) at 48 h post exposure, the biochemical alterations seen in gills were not present in digestive gland. The results indicate that gills are able to incorporate zinc prior (24 h) to digestive gland (48 h), leading to earlier mitochondrial disruption and oxidative stress. Our data suggest that gills are the initial target of ZnONP and that mitochondria are organelles particularly susceptible to ZnONP in C. gigas.