Responses of the desert green algae, Chlorella sp. to drought stress.

Desert algae are important components of the desert soil crust and play an essential role in desert soil ecosystem development. Owing to their special habitat, desert algae are often exposed to harsh environments, among which drought represents the most common stress. Green algae are considered to have drought tolerance potential; however, only a few studies have investigated this. In this study, we selected the green alga Chlorella sp., which was isolated from desert soil, and studied its physiological response to polyethylene glycol (PEG) 6000-induced drought stress. The results showed that drought stress can affect the photosynthetic efficiency of Chlorella sp., reduce its water retention ability, and destroy its ultrastructure. However, Chlorella sp. can cope with drought stress through a series of physiological regulatory strategies. Protective strategies include quick recovery of photosynthetic efficiency and increased chlorophyll content. In addition, induced synthesis of soluble proteins, lipids, and extracellular polysaccharide (EPS), and accumulation of osmotic regulatory substances, such as sucrose and trehalose, also contribute to improving drought tolerance in Chlorella sp. This study provides insights into the physiological responses of Chlorella sp. to drought stress, which may be valuable for understanding the underlying drought adaptation mechanisms of desert green algae.

Exploring cell aggregation as a defense strategy against perchlorate stress in Chlamydomonas reinhardtii through multi-omics analysis.

Perchlorate (ClO4-) is a type of novel, widely distributed, and persistent inorganic pollutant. However, the impacts of perchlorate on freshwater algae remain unclear. In this study, the response and defense mechanisms of microalgae (Chlamydomonas reinhardtii) under perchlorate stress were investigated by integrating physiological and biochemical monitoring, transcriptomics, and metabolomics. Weighted gene co-expression network analysis (WGCNA) of transcriptome data was used to analyze the relationship between genes and phenotype and screen the key pathways. C. reinhardtii exhibited aggregate behavior when exposed to 100- and 200-mM perchlorate but was restored to its unicellular lifestyle when transferred to fresh medium. WGCNA results found that the "carbohydrate metabolism" and "lipid metabolism" pathways were closely related to cell aggregation phenotype. The differential expression genes (DEGs) and differentially accumulated metabolites (DAMs) of these pathways were upregulated, indicating that the lipid and carbohydrate metabolisms were enhanced in aggregated cells. Additionally, most genes and metabolites related to phytohormone abscisic acid (ABA) biosynthesis and the mitogen-activated protein kinase (MAPK) signaling pathway were significantly upregulated, indicating their crucial roles in the signal transmission of aggregated cells. Meanwhile, in aggregated cells, extracellular polymeric substances (EPS) and lipid contents increased, photosynthesis activity decreased, and the antioxidant system was activated. These characteristics contributed to C. reinhardtii's improved resistance to perchlorate stress. Above results demonstrated that cell aggregation behavior was the principal defense strategy of C. reinhardtii against perchlorate. Overall, this study sheds new light on the impact mechanisms of perchlorate to aquatic microalgae and provides multi-omics insights into the research of multicellular-like aggregation as an adaptation strategy to abiotic stress. These results are beneficial for assessing the risk of perchlorate in aquatic environments.

UV-B radiation increased the sensitivity of Tibetan soil cyanobacterium Loriellopsis cavernicola to the herbicide glyphosate.

The high concentrations of herbicide and UV-B radiation are two stresses for Tibetan soil microorganisms, but there is limited information about the combined effects of herbicide and UV-B radiation on their levels of stress. In this study, the Tibetan soil cyanobacterium Loriellopsis cavernicola was used to investigate the combined inhibitory effect of the herbicide glyphosate and UV-B radiation on the cyanobacterial photosynthetic electron transport through an analysis of the photosynthetic activity, photosynthetic pigments, chlorophyll fluorescence and antioxidant system activity. The results demonstrated that treatment with herbicide or UV-B radiation and the combination of both stresses caused a decrease in the photosynthetic activity, interfered with the photosynthetic electron transport, and caused the accumulation of oxygen radicals and the degradation of photosynthetic pigments. In contrast, the combined treatment of glyphosate and UV-B radiation had a synergistic effect, i.e., the sensitivity of cyanobacteria to glyphosate increased in the presence of UV-B radiation, which caused the photosynthesis of cyanobacteria to have a greater impact. Since cyanobacteria are the primary producers of soil ecosystems, a high intensity of UV-B radiation in the plateau areas could enhance the inhibition of glyphosate on cyanobacteria, which could affect the ecological health and sustainable development of plateau soils.

Protection and Damage Repair Mechanisms Contributed To the Survival of Chroococcidiopsis sp. Exposed To a Mars-Like Near Space Environment.

Chroococcidiopsis spp. can withstand extremely harsh environments, including a Mars-like environment. However, studies are lacking on the molecular mechanisms of Chroococcidiopsis sp. surviving in Mars-like environments. In the HH-21-5 mission, the desert cyanobacterium Chroococcidiopsis sp. was exposed to a Mars-like environment (near space; 35 km altitude) for 4 h, and a single-factor environment of near space was simulated on the ground. We investigated the survival and endurance mechanisms of Chroococcidiopsis sp. ASB-02 after exposing it to near space by studying its physiological and transcriptional properties. After the exposure, Chroococcidiopsis sp. ASB-02 exhibited high cell viability, although photosystem II activity decreased and the levels of reactive oxygen species increased. The single-factor simulation experiments revealed that for the survival of Chroococcidiopsis sp. ASB-02 in near space, UV radiation was the most important limiting factor, and it was followed by temperature. The near space environment triggered multiple metabolic pathway responses in Chroococcidiopsis sp. ASB-02. The upregulation of extracellular polysaccharides as well as carotenoid and scytonemin biosynthesis genes in response to UV radiation attenuated the extent of radiation reaching the cells. At the same time, genes related to protein synthesis were upregulated in response to the low temperature, overcoming the decrease in metabolic activity that was caused by the low temperature. In near space and after rehydration, the genes involved in various DNA and photosystem II repair pathways were upregulated. This reflected the damage to the DNA and photosystem II protein subunits in cells during the flight and suggested that repair mechanisms play an important role in the recovery of Chroococcidiopsis sp. ASB-02. IMPORTANCE This study reported that the protective and repair mechanisms of Chroococcidiopsis sp. ASB-02 contributed to its endurance ability in a Mars-like near space environment. In Chroococcidiopsis sp. ASB-02, a Mars-like near space environment activated the expression of genes involved in extracellular polysaccharides (EPS), carotenoid, scytonemin, and protein syntheses, which provided additional protection. Additionally, the cell damage repair process enhanced the recovery rate of Chroococcidiopsis sp. ASB-02 after the flight. This study will help to enhance the understanding of the tolerance mechanism of Chroococcidiopsis sp. and to provide important guidance as to the survival requirements for microbial life in a Mars-like environment.

Cell damage repair mechanism in a desert green algae Chlorella sp. against UV-B radiation.

The protective ozone layer is continually depleting owing to an increase in the levels of solar UV-B radiation, which has harmful effects on organisms. Algae in desert soil can resist UV-B radiation, but most research on the radiation resistance of desert algae has focused on cyanobacteria. In this study, we found that desert green algae, Chlorella sp., could maintain high photosynthetic activity under UV-B stress. To examine the tolerance mechanism of the desert green algae photosystem, we observed the physiological and transcriptome-level responses of Chlorella sp. to high doses of UV-B radiation. The results showed that the reactive oxygen species (ROS) content first increased and then decreased, while the malondialdehyde (MDA) content revealed no notable lipid peroxidation during the UV-B exposure period. These results suggested that Chlorella sp. may have strong system characteristics for scavenging ROS. The antioxidant enzyme system showed efficient alternate coordination, which exhibited a protective effect against enhanced UV-B radiation. DNA damage and the chlorophyll and soluble protein contents had no significant changes in the early irradiation stage; UV-B radiation did not induce extracellular polysaccharides (EPS) synthesis. Transcriptomic data revealed that a strong photosynthetic system, efficient DNA repair, and changes in the expression of genes encoding ribosomal protein (which aid in protein synthesis and improve resistance) are responsible for the high UV-B tolerance characteristics of Chlorella sp. In contrast, EPS synthesis was not the main pathway for UV-B resistance. Our results revealed the potential cell damage repair mechanisms within Chlorella sp. that were associated with high intensity UV-B stress, thereby providing insights into the underlying regulatory adaptations of desert green algae.

Photosynthesis Responses of Tibetan Freshwater Algae Chlorella vulgaris to Herbicide Glyphosate.

With the development of agriculture and the widespread application of agrichemicals in Tibet, herbicide residues have become a threat to the ecological safety of Tibetan water bodies. Algae, as the producers in the food chain in water bodies, play an important role in aquatic ecosystems. Therefore, the impact of herbicides on Tibetan algae is of great significance for evaluating ecological health and the protection of Tibetan water ecosystems. In this study, we investigated the inhibitory effect of glyphosate, a herbicide, on the photosynthetic system of Chlorella vulgaris, Tibetan algae, by determining chlorophyll fluorescence and the activity of an antioxidant system. The results revealed that glyphosate at low concentration did not affect the photosynthetic activity of C. vulgaris; however, glyphosate at a high concentration significantly inhibited photosynthetic activity and reduced pigment content. Moreover, high levels of glyphosate also decreased photochemical efficiency and electron transport rate and resulted in ROS accumulation, high SOD activity, and lipid peroxidation. These results suggested that glyphosate could decrease the primary production of aquatic ecosystems and influence their performance. Therefore, reducing the herbicide levels could protect the Tibetan aquatic environment and maintain the health of ecosystems.

Exposure of cyanobacterium Nostoc sp. to the Mars-like stratosphere environment.

During the HH-19-2 flight mission of the Chinese Scientific Experimental System, dried Nostoc sp. cells were exposed to the stratosphere environment (32,508 m altitude) for 3 h and 22 min. The atmospheric pressure, temperature, relative humidity, and ionizing and non-ionizing radiation levels at that altitude are similar to those on the surface of Mars. Although analyses revealed decreased photosynthetic activity, a decline in autofluorescence, and damage to the cellular morphology in the flight-exposed sample, the death rate was low (28%). Physiological changes were not obvious after the exposure to the Mars-like vacuum conditions. The ground-exposed samples showed a similar trend to the flight-exposed samples, but the damage was relatively slight. RNA-sequencing data revealed a number of affected metabolic pathways: photosynthetic system and CO2 fixation function, activation of antioxidant systems, heat shock protein, DNA repair, and protein synthesis. Results suggest that Nostoc sp. has the potential to survive in a Mars-like environment and that it may be a suitable pioneer species to colonize Mars in the future in closed life-support systems (base) or in localities with relatively suitable conditions for life, such as localities with water available.

Carbon-negative and high-rate nutrient removal using mixotrophic microalgae.

Mixotrophic microalgae have demonstrated great potential for wastewater nutrient removal. How autotrophy/heterotrophy shares affect nutrient removal as well as carbon budget has not been understood. In this study, the autotrophy/heterotrophy shares in mixotrophy were quantified, and N removal rate and carbon budget under different mixotrophic autotrophy/heterotrophy shares were modeled. The results showed that mixotrophic N removal rate reached 2.09 mg L-1h-1, which was 53.18% and 37.98% higher than removal rates in autotrophic (0.97 mg L-1h-1) and heterotrophic (1.25 mg L-1h-1) controls. Mixotrophic-autotrophy and mixotrophic-heterotrophy contributed 1.15 mg L-1h-1 and 0.94 mg L-1h-1 in N removal, respectively. Model disclosed that at balanced share of 6:4, more than 2 mg L-1h-1N removal could be achieved, similar to bacterial nitrogen removal rate but with a negative carbon budget of 6.21 mg L-1h-1. Nutrient removal using mixotrophic microalgae would lead to carbon negative sustainable wastewater treatment and resource recycling.

Survival of desert algae Chlorella exposed to Mars-like near space environment.

Desert was considered terrestrial analogues of Mars. In this study, dried cells of desert green algae Chlorella were exposed to Mars-like near-space environment using high-altitude scientific balloons. We found that while a majority of Chlorella cells survived, they exhibited considerable damage, such as low photosynthetic activity, reduced cell growth, increased cell mortality rate, and altered chloroplast and mitochondrial ultrastructure. Additionally, transcriptome analysis of near space-exposed Chlorella cells revealed 3292 differentially expressed genes compared to cells in the control ground group, including heat shock proteins, antioxidant enzymes, DNA repair systems, as well as proteins related to the PSII apparatus and ribosomes. These data shed light on the possible survival strategy of desert algae to near space environments. Our results indicated that Mars-like near space conditions represent an extreme environment for desert algae in terms of temperature, pressure, and radiations. The survival strategy of Chlorella in response to near space will help gain insights into the possibility of extremophile colonization on the surface of Mars and in similar extraterrestrial habitats.

Enhancement of DNA damage repair potential in germ cells of Caenorhabditis elegans by a volatile signal from their irradiated partners.

Radiation-induced bystander effects have been demonstrated within organisms. Recently, it is found that the organisms can also signal irradiation cues to their co-cultured partners in a waterborne manner. In contrast, there is a limited understanding of radiation-induced airborne signaling between individuals, especially on the aspect of DNA damage responses (DDR). Here, we establish a co-culture experimental system using Caenorhabdis elegans in a top-bottom layout, where communication between "top" and "bottom" worms is airborne. The radiation response of top worms is evaluated using radio-adaptive response (RAR) of embryonic lethality (F1), which reflects an enhancement in repair potential of germ cells to subsequent DNA damage. It is shown that gamma-irradiation of bottom worms alleviates the embryonic lethality of top worms caused by 25 Gy of subsequent gamma-irradiation, i.e. RAR, indicating that a volatile signal might play an essential role in radiation-induced inter-worm communication. The RAR is absent in the top worms impaired in DNA damage checkpoint, nucleotide excision repair, and olfactory sensory neurons, respectively. The induction of RAR is restricted to the mitotic zone of the female germline of hermaphrodites. These results indicate that the top worms sense the volatile signal through cephalic sensory neurons, and the neural stimulation distantly modulates the DDR in germ mitotic cells, leading to the enhancement of DNA damage repair potential. The volatile signal is produced specifically by the L3-stage bottom worms and functionally distinct from the known sex pheromone. Its production and/or release are regulated by water-soluble ascaroside pheromones in a population-dependent manner.

A genomics approach reveals the global genetic polymorphism, structure, and functional diversity of ten accessions of the marine model diatom Phaeodactylum tricornutum.

Diatoms emerged in the Mesozoic period and presently constitute one of the main primary producers in the world's ocean and are of a major economic importance. In the current study, using whole genome sequencing of ten accessions of the model diatom Phaeodactylum tricornutum, sampled at broad geospatial and temporal scales, we draw a comprehensive landscape of the genomic diversity within the species. We describe strong genetic subdivisions of the accessions into four genetic clades (A-D) with constituent populations of each clade possessing a conserved genetic and functional makeup, likely a consequence of the limited dispersal of P. tricornutum in the open ocean. We further suggest dominance of asexual reproduction across all the populations, as implied by high linkage disequilibrium. Finally, we show limited yet compelling signatures of genetic and functional convergence inducing changes in the selection pressure on many genes and metabolic pathways. We propose these findings to have significant implications for understanding the genetic structure of diatom populations in nature and provide a framework to assess the genomic underpinnings of their ecological success and impact on aquatic ecosystems where they play a major role. Our work provides valuable resources for functional genomics and for exploiting the biotechnological potential of this model diatom species.

Foxtail millet (Setaria italica (L.) P. Beauv) CIPKs are responsive to ABA and abiotic stresses.

CBL-interacting protein kinases (CIPKs) have been shown to regulate a variety of environmental stress-related signalling pathways in plants. Foxtail millet (Setaria italica (L.) P. Beauv) is known worldwide as a relatively stress-tolerant C4 crop species. Although the foxtail millet genome sequence has been released, little is known about the functions of CIPKs in foxtail millet. Therefore, a systematic genome-wide analysis of CIPK genes in foxtail millet was performed. In total, 35 CIPK members were identified in foxtail millet and divided into four subgroups (I to IV) on the basis of their phylogenetic relationships. Phylogenetic and gene structure analyses clearly divided all SiCIPKs into intron-poor and intron-rich clades. Cis-element analysis subsequently indicated that these SiCIPKs may be involved in responses to abiotic stimuli, hormones, and light signalling during plant growth and development, and stress-induced expression profile analysis revealed that all the SiCIPKs are involved in various stress signalling pathways. These results suggest that the CIPK genes in foxtail millet exhibit the basic characteristics of CIPK family members and play important roles in response to abiotic stresses. The results of this study will contribute to future functional characterization of abiotic stress responses mediated by CIPKs in foxtail millet.

Negative Modulation of Bystander DNA Repair Potential by X-Ray Targeted Tissue Volume in Arabidopsis thaliana.

Radiation-induced bystander effects (RIBE) entail a cascade of bystander signals produced by the hit cells to the neighboring cells to regulate various biological processes including DNA damage repair. However, there is little clarity regarding the effect of radiation-targeted volume (hit cell amount) on the DNA repair potential of the bystander cells. This is especially important to understand in the context of the whole organism, where the target usually consists of multiple types of cells/tissues. To address this question, model plant Arabidopsis thaliana was locally irradiated, and the DNA repair potential of bystander root-tip cells was assessed based on their radioresistance to subsequent high-dose radiation, i.e. radioadaptive responses (RAR). We found that X-ray irradiation of the aerial parts (AP) of A. thaliana seedlings (5 Gy) initiated RAR in the root-tip cells, which exhibited an alleviated repression of root growth and root cell division, and reduced amount of DNA strand breaks. We also observed an improvement in the repair efficiency of the homologous recombination (HR) and non-homologous end joining (NHEJ) pathways in the bystander root tip cells. We further expanded the X-ray targeted volume to include the aerial parts with upper parts of the primary root and compared it with X-ray irradiated aerial parts alone. Comparative analysis revealed that RAR for these end points either disappeared or decreased; specifically, the repair efficiency of HR was significantly reduced, indicating that radiation-targeted volume negatively modulates the bystander DNA repair potential. In contrast, X-ray irradiation of upper part of the primary root alone did not induce RAR of the root tip cells. Thus, we propose that additional X-ray irradiation of upper part of the primary root reduces the bystander DNA repair potential, possibly by selectively disturbing the transport of bystander signals responsible for HR repair.

Cre-miR914-regulated RPL18 is involved with UV-B adaptation in Chlamydomonas reinhardtii.

UV radiation is a serious threat to life, and algae have developed highly efficient adaptations to UV radiation through the course of evolution. To date, studies investigating the mechanisms of UV adaptation in algae have focused on physiological regulation and associated protein coding genes, with only a few reports on associated protein non-coding genes. In a previous study, we found that Cre-miR914 was significantly down-regulated in Chlamydomonas reinhardtii in response to heat shock. In the present study, we aimed to determine whether Cre-miR914 plays a role in response to UV-B radiation. Our bioinformatics analysis indicated that the potential target gene of Cre-miR914 is ribosomal protein L18 (RPL18). We also measured the expression of Cre-miR914 and RPL18 in response to UV-B radiation through qPCR analysis. Then, we constructed cell lines overexpressing Cre-miR914 or RPL18, and performed survival experiments under UV-B stress. The results showed that Cre-miR914 overexpression decreased resistance while RPL18 overexpression enhanced tolerance to UV-B radiation. These results indicate that Cre-miR914 and its potential target gene RPL18 are involved in the adaptation to UV-B in C. reinhardtii.

Cell damage caused by ultraviolet B radiation in the desert cyanobacterium Phormidium tenue and its recovery process.

Phormidium tenue, a cyanobacterium that grows in the topsoil of biological soil crusts (BSCs), has the highest recovery rate among desert crust cyanobacteria after exposure to ultraviolet B (UV-B) radiation. However, the mechanism underlying its recovery process is unclear. To address this issue, we measured chlorophyll a fluorescence, generation of reactive oxygen species (ROS), lipid peroxidation, and repair of DNA breakage in P. tenue following exposure to UV-B. We found that UV-B radiation at all doses tested reduced photosynthesis and induced cell damage in P. tenue. However, P. tenue responded to UV-B radiation by rapidly reducing photosynthetic activity, which protects the cell by leaking less ROS. Antioxidant enzymes, DNA damage repair systems, and UV absorbing pigments were then induced to mitigate the damage caused by UV-B radiation. The addition of exogenous antioxidant chemicals ascorbate and N-acetylcysteine also mitigated the harmful effects caused by UV-B radiation and enhanced the recovery process. These chemicals could aid in the resistance of P. tenue to the exposure of intense UV-B radiation in desertified areas when inoculated onto the sand surface to form artificial algal crusts.

MicroRNAs modulate adaption to multiple abiotic stresses in Chlamydomonas reinhardtii.

MicroRNAs play an important role in abiotic stress responses in higher plants and animals, but their role in stress adaptation in algae remains unknown. In this study, the expression of identified and putative miRNAs in Chlamydomonas reinhardtii was assessed using quantitative polymerase chain reaction; some of the miRNAs (Cre-miR906-3p) were up-regulated, whereas others (Cre-miR910) were down-regulated when the species was subjected to multiple abiotic stresses. With degradome sequencing data, we also identified ATP4 (the d-subunit of ATP synthase) and NCR2 (NADPH: cytochrome P450 reductase) as one of the several targets of Cre-miR906-3p and Cre-miR910, respectively. Q-PCR data indicated that ATP4, which was expressed inversely in relation to Cre-miR906-3p under stress conditions. Overexpressing of Cre-miR906-3p enhanced resistance to multiple stresses; conversely, overexpressing of ATP4 produced the opposite effect. These data of Q-PCR, degradome sequencing and adaptation of overexpressing lines indicated that Cre-miR906-3p and its target ATP4 were a part of the same pathway for stress adaptation. We found that Cre-miR910 and its target NCR2 were also a part of this pathway. Overexpressing of Cre-miR910 decreased, whereas that of NCR2 increased the adaption to multiple stresses. Our findings suggest that the two classes of miRNAs synergistically mediate stress adaptation in algae.

Surface modification of polytetrafluoroethylene column for two-stationary phase separations by counter-current chromatography.

To improve the separation capability of CCC, a novel solid-liquid two-stationary phases CCC (ASP-CCC) column was prepared employing graphene oxide (GO) conjugated poly-dopamine (PD) coating (GO/PD) as auxiliary stationary phase (ASP). The results of Scanning electron microscopy (SEM), contact angle and X-ray photoelectron spectroscopy (XPS) indicated that nanostructured GO and PD were successfully grafted on the inner wall of the PTFE column. Three alkaloid compounds were selected as the target analytes to evaluate the performance of the novel column. Because of the intermolecular force (hydrogen bond, electrostatic interaction and π-π interaction) between the ASP and model compounds, three analytes were well separated with this novel ASP-CCC column. Additionally, the novel column exhibited higher stationary phase retention ratio, about 8%, than original column without changing the chromatographic condition. Furthermore, the eluotropic sequence of analytes on novel column was in accordance with that in the original column. This suggested that the novel column is a CCC column with auxiliary stationary phase (ASP) in its own right, and the present separation mode is the combination of partition chromatography and adsorption chromatography.

Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii.

The unicellular green algae Chlamydomonas reinhardtii is a classic model for the study of flagella/cilia and photosynthesis, and it has recently been exploited for producing biopharmaceuticals and biofuel. Due to the low frequency of homologous recombination, reverse genetic manipulation in Chlamydomonas relies mainly on miRNA- and siRNA-based knockdown methods. However, the difficulty in constructing artificial miRNA vectors, laborious screening of knockdown transformants, and undesired epigenetic silencing of exogenous miRNA constructs limit their application. We have established a one-step procedure to construct an artificial miRNA precursor by annealing eight oligonucleotides of approximately 40 nucleotides. In the final construct, the Gaussia princeps luciferase gene (G-Luc) is positioned between the promoter and the artificial miRNA precursor so that knockdown strains may quickly be screened by visualizing luciferase luminescence using a photon-counting camera. Furthermore, the luciferase activity of transformants correlates with the knockdown level of two test target proteins: the chloroplast protein VIPP1 (vesicle inducing protein in plastids 1) and the flagellar protein CDPK3 (calcium-dependent protein kinase 3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit 2) to the miRNA construct enhanced both the luciferase activity and the miRNA knockdown efficiency. A second miRNA vector incorporated the promoter of the nitrate reductase gene to allow inducible expression of the artificial miRNA. These vectors will facilitate application of the artificial miRNA and provide tools for studying the mechanism of epigenetics in Chlamydomonas, and may also be adapted for use in other model organisms.

Reproductive and locomotory capacities of Caenorhabditis elegans were not affected by simulated variable gravities and spaceflight during the Shenzhou-8 mission.

Reproduction and locomotion are essential features of animals that help to facilitate their interaction with the surrounding environment. Previous studies have produced inconsistent results on behavioral response to spaceflight by the model animal Caenorhabditis elegans (C. elegans) in liquid culture. Using standard agar-based nematode growth medium (NGM), we show here that both reproductive and locomotory capacities of C. elegans were not significantly changed by centrifuge-produced hypergravity or clinostat-simulated microgravity. To investigate the effect of actual spaceflight on C. elegans, a nematode test unit was specifically designed to maintain its normal growth on solid NGM slides and to allow automatic RNA fixation on board the Shenzhou-8 spaceflight. We did not detect alteration in either brood size of immediate progenies from postflight nematodes or locomotory behavior, including speed of locomotion, frequency of reversals, and rate of body bends of space-flown nematodes collected directly from nematode test units. Our results provide clear evidence that the nematode test unit is an appropriate apparatus for nematode growth on standard NGM and can be used for on-orbit analysis of C. elegans, including onboard RNA fixation for molecular analysis and real-time video acquisition for behavioral analysis, which are critical for further studies in unmanned spaceflight and outer space exploration.

Zebrafish neurotoxicity from aphantoxins--cyanobacterial paralytic shellfish poisons (PSPs) from Aphanizomenon flos-aquae DC-1.

Aphanizomenon flos-aquae (A. flos-aquae), a cyanobacterium frequently encountered in water blooms worldwide, is source of neurotoxins known as PSPs or aphantoxins that present a major threat to the environment and to human health. Although the molecular mechanism of PSP action is well known, many unresolved questions remain concerning its mechanisms of toxicity. Aphantoxins purified from a natural isolate of A. flos-aquae DC-1 were analyzed by high-performance liquid chromatography (HPLC), the major component toxins were the gonyautoxins1 and 5 (GTX1 and GTX5, 34.04% and 21.28%, respectively) and the neosaxitoxin (neoSTX, 12.77%). The LD50 of the aphantoxin preparation was determined to be 11.33 µg/kg (7.75 µg saxitoxin equivalents (STXeq) per kg) following intraperitoneal injection of zebrafish (Danio rerio). To address the neurotoxicology of the aphantoxin preparation, zebrafish were injected with low and high sublethal doses of A. flos-aquae DC-1 toxins 7.73 and 9.28 µg /kg (5.3 and 6.4 µg STXeq/kg, respectively) and brain tissues were analyzed by electron microscopy and RT-PCR at different timepoints postinjection. Low-dose aphantoxin exposure was associated with chromatin condensation, cell-membrane blebbing, and the appearance of apoptotic bodies. High-dose exposure was associated with cytoplasmic vacuolization, mitochondrial swelling, and expansion of the endoplasmic reticulum. At early timepoints (3 h) many cells exhibited characteristic features of both apoptosis and necrosis. At later timepoints apoptosis appeared to predominate in the low-dose group, whereas necrosis predominated in the high-dose group. RT-PCR revealed that mRNA levels of the apoptosis-related genes encoding p53, Bax, caspase-3, and c-Jun were upregulated after aphantoxin exposure, but there was no evidence of DNA laddering; apoptosis could take place by pathways independent of DNA fragmentation. These results demonstrate that aphantoxin exposure can cause cell death in zebrafish brain tissue, with low doses inducing apoptosis and higher doses inducing necrosis.