Application of heavy-ion-beam irradiation to breeding large rotifer.

In larviculture facilities, rotifers are generally used as an initial food source, while a proper size of live feeds to connect rotifer and Artemia associated with fish larval growth is needed. The improper management of feed size and density induces mass mortality and abnormal development of fish larvae. To improve the survival and growth of target larvae, this study applied carbon and argon heavy-ion-beam irradiation in mutation breeding to select rotifer mutants with larger lorica sizes. The optimal irradiation conditions of heavy-ion beam were determined with lethality, reproductivity, mutant frequency, and morphometric characteristics. Among 56 large mutants, TYC78, TYC176, and TYA41 also showed active population growth. In conclusion, (1) heavy-ion-beam irradiation was defined as an efficient tool for mutagenesis of rotifers and (2) the aforementioned 3 lines that have larger lorica length and active population growth may be used as a countermeasure of live feed size gap during fish larviculcure.

Mutation accumulation in populations of varying size: large effect mutations cause most mutational decline in the rotifer Brachionus calyciflorus under UV-C radiation.

Theory predicts that fitness decline via mutation accumulation will depend on population size, but there are only a few direct tests of this key idea. To gain a qualitative understanding of the fitness effect of new mutations, we performed a mutation accumulation experiment with the facultative sexual rotifer Brachionus calyciflorus at six different population sizes under UV-C radiation. Lifetime reproduction assays conducted after ten and sixteen UV-C radiations showed that while small populations lost fitness, fitness losses diminished rapidly with increasing population size. Populations kept as low as 10 individuals were able to maintain fitness close to the nonmutagenized populations throughout the experiment indicating that selection was able to remove the majority of large effect mutations in small populations. Although our results also seem to imply that small populations are effectively immune to mutational decay, we caution against this interpretation. Given sufficient time, populations of moderate to large size can experience declines in fitness from accumulating weakly deleterious mutations as demonstrated by fitness estimates from simulations and, tentatively, from a long-term experiment with populations of moderate size. There is mounting evidence to suggest that mutational distributions contain a heavier tail of large effects. Our results suggest that this is also true when the mutational spectrum is altered by UV radiation.

Tolerance to X-rays and Heavy Ions (Fe, He) in the Tardigrade Richtersius coronifer and the Bdelloid Rotifer Mniobia russeola.

The aim of this study was to analyze tolerance to heavy ions in desiccated animals of the eutardigrade Richtersius coronifer and the bdelloid rotifer Mniobia russeola within the STARLIFE project. Both species were exposed to iron (Fe) and helium (He) ions at the Heavy Ion Medical Accelerator in Chiba (HIMAC) in Chiba, Japan, and to X-rays at the German Aerospace Center (DLR) in Cologne, Germany. Results show no effect of Fe and He on viability up to 7 days post-rehydration in both R. coronifer and M. russeola, while X-rays tended to reduce viability in R. coronifer at the highest doses. Mean egg production rate tended to decline with higher doses in R. coronifer for all radiation types, but the pattern was not statistically confirmed. In M. russeola, there was no such tendency for a dose response in egg production rate. These results confirm the previously reported high tolerance to high linear energy transfer (LET) radiation in tardigrades and show for the first time that bdelloid rotifers are also very tolerant to high-LET radiation. These animal phyla represent the most desiccation- and radiation-tolerant animals on Earth and provide excellent eukaryotic models for astrobiological research. Key Words: Tardigrada-Rotifera-Radiation tolerance-Heavy ions-X-rays. Astrobiology 17, 163-167.

Protein damage, radiation sensitivity and aging.

This paper promotes a concept that protein damage determines radiation resistance and underlies aging and age-related diseases. The first bottleneck in cell recovery from radiation damage is functional (proteome) rather than informational (DNA), since prokaryotic and eukaryotic cell death correlates with incurred protein, but not DNA, damage. Proteome protection against oxidative damage determines survival after ionizing or UV irradiation, since sufficient residual proteome activity is required to turn on the DNA damage response activating DNA repair and protein renewal processes. Extreme radiation and desiccation resistance of rare bacterial and animal species is accounted for by exceptional constitutive proteome protection against oxidative damage. After excessive radiation their well-protected proteome faithfully reconstitutes a transcription-competent genome from hundreds of DNA fragments. The observation that oxidative damage targeted selectively to cellular proteins results in aging-like phenotypes suggests that aging and age-related diseases could be phenotypic consequences of proteome damage patterns progressing with age.

Acute Toxicity of Gamma Radiation to the Monogonont Rotifer Brachionus koreanus.

We examined the tolerance of the monogonont rotifer Brachionus koreanus in response to gamma radiation. In order to determine the median lethal dose (LD50) of rotifers against gamma radiation, we irradiated B. koreanus with gamma rays from 0 to 7000 grays (Gy). The LD50s were 2900 and 2300 Gy at 24 h (LD50-24 h) and 96 h (LD50-96 h) after irradiation, respectively. In addition, the no observed effect levels (NOEL) were 1500 and 1000 Gy at 24 and 96 h, respectively. This is the first determination of lethal doses of gamma radiation for B. koreanus, which could be useful in ecological assessment of gamma radiation toward aquatic life and could be useful for understanding toxic mechanisms over sublethal doses.

Rotifer dynamics in three shallow lakes from the Salado river watershed (Argentina): the potential modulating role of incident solar radiation.

In turbid Pampean lakes, incident solar radiation is a major driver of plankton seasonal dynamics. Higher light availability in summer translates into higher primary production, and therefore more food for zooplankton grazers. However, experimental evidence suggests that food produced under the high irradiance conditions prevailing in summer are less suitable to sustain rotifer population growth than that produced under the lower irradiance conditions typical of winter. Here, we analysed time series datasets corresponding to three shallow lakes from the Salado river watershed. This analysis provided evidence for similar seasonal patterns of rotifer relative abundance over a large geographic area. In addition, we performed life table experiments to test the hypothesis that natural seston produced in winter could sustain higher population growth rates than seston produced in summer. We suggest that the natural seasonal changes in temperature and food generate successive time windows, which may be capitalized by the different grazer species, resulting in predictable phenology of grazer populations.

Gateway to genetic exchange? DNA double-strand breaks in the bdelloid rotifer Adineta vaga submitted to desiccation.

The bdelloid rotifer lineage Adineta vaga inhabits temporary habitats subjected to frequent episodes of drought. The recently published draft sequence of the genome of A. vaga revealed a peculiar genomic structure incompatible with meiosis and suggesting that DNA damage induced by desiccation may have reshaped the genomic structure of these organisms. However, the causative link between DNA damage and desiccation has never been proven to date in rotifers. To test for the hypothesis that desiccation induces DNA double-strand breaks (DSBs), we developed a protocol allowing a high survival rate of desiccated A. vaga. Using pulsed-field gel electrophoresis to monitor genomic integrity, we followed the occurrence of DSBs in dried bdelloids and observed an accumulation of these breaks with time spent in dehydrated state. These DSBs are gradually repaired upon rehydration. Even when the genome was entirely shattered into small DNA fragments by proton radiation, A. vaga individuals were able to efficiently recover from desiccation and repair a large amount of DSBs. Interestingly, when investigating the influence of UV-A and UV-B exposure on the genomic integrity of desiccated bdelloids, we observed that these natural radiations also caused important DNA DSBs, suggesting that the genome is not protected during the desiccated stage but that the repair mechanisms are extremely efficient in these intriguing organisms.

Sublethal gamma irradiation affects reproductive impairment and elevates antioxidant enzyme and DNA repair activities in the monogonont rotifer Brachionus koreanus.

To examine the effects of gamma radiation on marine organisms, we irradiated several doses of gamma ray to the microzooplankton Brachionus koreanus, and measured in vivo and in vitro endpoints including the survival rate, lifespan, fecundity, population growth, gamma ray-induced oxidative stress, and modulated patterns of enzyme activities and gene expressions after DNA damage. After gamma radiation, no individuals showed any mortality within 96 h even at a high intensity (1200 Gy). However, a reduced fecundity (e.g. cumulated number of offspring) of B. koreanus at over 150 Gy was observed along with a slight decrease in lifespan. At 150 Gy and 200 Gy, the reduced fecundity of the rotifers led to a significant decrease in population growth, although in the second generation the population growth pattern was not affected even at 200 Gy when compared to the control group. At sub-lethal doses, reactive oxygen species (ROS) levels dose-dependently increased with GST enzyme activity. In addition, up-regulations of the antioxidant and chaperoning genes in response to gamma radiation were able to recover cellular damages, and life table parameters were significantly influenced, particularly with regard to fecundity. DNA repair-associated genes showed significantly up-regulated expression patterns in response to sublethal doses (150 and 200 Gy), as shown in the expression of the gamma-irradiated B. koreanus p53 gene, suggesting that these sublethal doses were not significantly fatal to B. koreanus but induced DNA damages leading to a decrease of the population size.

Identification of three doublesex genes in the monogonont rotifer Brachionus koreanus and their transcriptional responses to environmental stressor-triggered population growth retardation.

Doublesex and Mab-3-related transcription factor (Dmrt) gene family members have rarely been identified or characterized in aquatic invertebrates. In this study, we identified and characterized three DMdomain-containing genes - Dmrt11E, Dmrt93B, and Dmrt99B - in the monogonont rotifer, Brachionus koreanus. DMdomains of the proteins encoded by the B.koreanus Dmrt (Bk-Dmrt) genes had high similarities to DM domains of other invertebrate species. To understand the potential effects of environmental stressors on the transcriptional expression of Dmrt genes in rotifers, we exposed B.koreanus to a wide range of UV-B radiation and different concentrations of benzo[a]pyrene (B[a]P) over different time courses. Transcript levels of all Bk-Dmrt genes decreased significantly in response to relatively high doses of UV-B irradiation, and were also downregulated in response to exposure to UV-B radiation over time. Transcript levels of all Bk-Dmrt genes were downregulated in response to B[a]P exposure for 24h. This decrease in expression of all Bk-Dmrt genes was concomitant with the growth retardation induced by UV-B and B[a]P exposure. We concluded that both environmental stressors have detrimental effects on transcriptional regulation of all Bk-Dmrt genes, especially relatively high doses of these stressors, leading to growth retardation. However, further studies are required to better understand the potential role of Dmrt genes in environmental stressor-triggered growth retardation in the rotifer B.koreanus.

How does the 'ancient' asexual Philodina roseola (Rotifera: Bdelloidea) handle potential UVB-induced mutations?

Like other obligate asexuals, bdelloid rotifers are expected to suffer from degradation of their genomes through processes including the accumulation of deleterious mutations. However, sequence-based analyses in this regard remain inconclusive. Instead of looking for historical footprints of mutations in these ancient asexuals, we directly examined the susceptibility and ability to repair point mutations by the bdelloid Philodina roseola by inducing cyclobutane-pyrimidine dimers (CPDs) via exposure to UVB radiation (280-320 nm). For comparison, we performed analogous experiments with the facultative asexual monogonont rotifer Brachionus rubens. Different strategies were found for the two species. Philodina roseola appeared to shield itself from CPD induction through uncharacterized UV-absorbing compounds and, except for the genome reconstruction that occurs after desiccation, was largely unable to repair UVB-induced damage. By contrast, B. rubens was more susceptible to UVB irradiation, but could repair all induced damage in ~2 h. In addition, whereas UV irradiation had a significant negative impact on the reproductive output of P. roseola, and especially so after desiccation, that of B. rubens was unaffected. Although the strategy of P. roseola might suffice under natural conditions where UVB irradiation is less intense, the lack of any immediate CPD repair mechanisms in this species remains perplexing. It remains to be investigated how typical these results are for bdelloids as a group and therefore how reliant these animals are on desiccation-dependent genome repair to correct potential DNA damage given their obligate asexual lifestyle.

Extreme anti-oxidant protection against ionizing radiation in bdelloid rotifers.

Bdelloid rotifers, a class of freshwater invertebrates, are extraordinarily resistant to ionizing radiation (IR). Their radioresistance is not caused by reduced susceptibility to DNA double-strand breakage for IR makes double-strand breaks (DSBs) in bdelloids with essentially the same efficiency as in other species, regardless of radiosensitivity. Instead, we find that the bdelloid Adineta vaga is far more resistant to IR-induced protein carbonylation than is the much more radiosensitive nematode Caenorhabditis elegans. In both species, the dose-response for protein carbonylation parallels that for fecundity reduction, manifested as embryonic death. We conclude that the great radioresistance of bdelloid rotifers is a consequence of an unusually effective system of anti-oxidant protection of cellular constituents, including those required for DSB repair, allowing bdelloids to recover and continue reproducing after doses of IR causing hundreds of DSBs per nucleus. Bdelloid rotifers therefore offer an advantageous system for investigation of enhanced anti-oxidant protection and its consequences in animal systems.

Ultraviolet B retards growth, induces oxidative stress, and modulates DNA repair-related gene and heat shock protein gene expression in the monogonont rotifer, Brachionus sp.

Ultraviolet B (UV-B) radiation causes direct cellular damage by breakage of DNA strands and oxidative stress induction in aquatic organisms. To understand the effect of UV-B radiation on the rotifer, Brachionus sp., several parameters including 24-h survival rate, population growth rate, and ROS level were measured after exposure to a wide range of UV-B doses. To check the expression of other important inducible genes such as replication protein A (RPA), DNA-dependent protein kinase (DNA-PK), Ku70, Ku80, and heat shock proteins (hsps) after UV-B radiation, we observed dose- and time-dependency at 2kJ/m(2). We also examined 13 hsp genes for their roles in the UV-B damaged rotifer. Results showed that UV-B remarkably inhibited the population growth of Brachionus sp. The level of intracellular reactive oxygen species (ROS) was high at 2kJ/m(2), suggesting that 2kJ/m(2) would already be toxic. This result was supported by other enzymatic activities, such as GSH levels, glutathione peroxidase, glutathione S-transferase, and glutathione reductase. For dose dependency, low doses of UV-B radiation (2, 4, and 6kJ/m(2)) significantly up-regulated the examined genes (e.g. RPA, DNA-PK, Ku70, and Ku80). For the time course study, RPA genes showed immediate up-regulation but returned to basal or lower expression levels compared to the control 3h after UV-B exposure. The DNA-PK and Ku70/80 genes significantly increased, indicating that they may be involved in repairing processes against a low dose of UV-B exposure (2kJ/m(2)). At the basal level, the hsp90α1 gene showed the highest expression, and followed by hsp10, hsp30, hsp60, and hsc70, and hsp90ß in adults (w/o egg). In eggs, the hsp10 gene was expressed the highest, and followed by hsp30, hsp27, hsp90α1, and hsp60 genes. In real-time RT-PCR array on rotifer hsp genes, low doses of UV-B radiation (2 and 4kJ/m(2)) showed up-regulation of several hsp genes but most of the hsp genes showed down-regulation at 8kJ/m(2) and higher, indicating that significant Hsp-mediated cellular damage already occurred at low doses. For the time course study of four hsp genes (hsp20, hsp27, hsp70, hsp90α1), they showed a significant correlation for UV-B radiation (2kJ/m(2)). In this paper, we demonstrated that UV-B radiation would affect growth retardation with up- or down-regulation of some important genes in DNA replication, repair process, and chaperoning. This finding provides a better understanding of molecular mechanisms involved in UV-B-mediated cellular damage in the rotifer, Brachionus sp.

Effects of acute gamma-irradiation on community structure of the aquatic microbial microcosm.

To characterise indirect effects of ionising radiation on aquatic microbial communities, effects of acute gamma-irradiation were investigated in a microcosm consisting of populations of green algae (Chlorella sp. and Scenedesmus sp.) and a blue-green alga (Tolypothrix sp.) as producer; a ciliate protozoan (Cyclidium glaucoma), rotifers (Lecane sp. and Philodina sp.) and an oligochaete (Aeolosoma hemprichi) as consumer; and more than four species of bacteria as decomposers. Population changes in the constituent organisms were observed over 160 days after irradiation. Prokaryotic community structure was also examined by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Principle response curve analysis revealed that the populations of the microcosm as a whole were not significantly affected at 100Gy while they were adversely affected at 500-5000Gy in a dose-dependent manner. However, some effects on each population, including each bacterial population detected by DGGE, did not depend on radiation doses, and some populations in the irradiated microcosm were larger than those of the control. These unexpected results are regarded as indirect effects through interspecies interactions, and possible mechanisms are proposed originating from population changes in other organisms co-existing in the microcosm. For example, some indirect effects on consumers and decomposers likely arose from interspecies competition within each trophic level. It is also likely that prey-predator relationships between producers and consumers caused some indirect effects on producers.

Effects of acute gamma-irradiation on the aquatic microbial microcosm in comparison with chemicals.

Effects of acute gamma-irradiation were investigated in the aquatic microcosm consisting of green algae (Chlorella sp. and Scenedesmus sp.) and a blue-green alga (Tolypothrix sp.) as producers; an oligochaete (Aeolosoma hemprichi), rotifers (Lecane sp. and Philodina sp.) and a ciliate protozoan (Cyclidium glaucoma) as consumers; and more than four species of bacteria as decomposers. At 100 Gy, populations were not affected in any taxa. At 500-5000 Gy, one or three taxa died out and populations of two or three taxa decreased over time, while that of Tolypothrix sp. increased. This Tolypothrix sp. increase was likely an indirect effect due to interspecies interactions. The principal response curve analysis revealed that the main trend of the effects was a dose-dependent population decrease. For a better understanding of radiation risks in aquatic microbial communities, effect doses of gamma-rays compared with copper, herbicides and detergents were evaluated using the radiochemoecological conceptual model and the effect index for microcosm.

Extreme resistance of bdelloid rotifers to ionizing radiation.

Rotifers of class Bdelloidea are common invertebrate animals with highly unusual characteristics, including apparently obligate asexuality, the ability to resume reproduction after desiccation at any life stage, and a paucity of transposable genetic elements of types not prone to horizontal transmission. We find that bdelloids are also extraordinarily resistant to ionizing radiation (IR). Reproduction of the bdelloids Adineta vaga and Philodina roseola is much more resistant to IR than that of Euchlanis dilatata, a rotifer belonging to the desiccation-intolerant and facultatively sexual class Monogononta, and all other animals for which we have found relevant data. By analogy with the desiccation- and radiation-resistant bacterium Deinococcus radiodurans, we suggest that the extraordinary radiation resistance of bdelloid rotifers is a consequence of their evolutionary adaptation to survive episodes of desiccation encountered in their characteristic habitats and that the damage incurred in such episodes includes DNA breakage that is repaired upon rehydration. Such breakage and repair may have maintained bdelloid chromosomes as colinear pairs and kept the load of transposable genetic elements low and may also have contributed to the success of bdelloid rotifers in avoiding the early extinction suffered by most asexuals.

Effects of U.V. radiation on the lifespan of the rotifer Asplanchna brightwelli.

Groups of rotifers of the species Asplanchna brightwelli were exposed to U.V. irradiation at dosages ranging from 50 to 4800 J/m2. The lifespan of the rotifers was significantly reduced by exposure to U.V. in the range of 200 to 4800 J/m2. A logarithmic decline in lifespan was seen as the U.V. dose increased. Rotifers were most sensitive to U.V. exposure during the prereproductive stage at the beginning of their lifecycle. Exposure of rotifers to visible light following U.V. radiation provided no evidence that photoreactivation could influence lifespan in this species.