The combined effects of salinity, hypoxia, and oil exposure on survival and gene expression in developing sheepshead minnows, Cyprinodon variegatus.

The 2010 Deepwater Horizon oil spill released approximately 780 million liters of crude oil contaminating coastal habitats from Texas to Florida which are important habitats for many fish species during early life stages. These diverse habitats are also prone to rapid fluctuations in water quality, such as dissolved oxygen concentration and salinity. The consequence of combined exposure to crude oil and suboptimal environmental conditions during early life stage development of fish is still largely unknown. The objective of this project was to investigate the impacts of exposure to crude oil in combination with varying environmental stressors on developing Cyprinodon variegatus survival, growth, and gene expression. Three life stages (embryonic, post-hatch, and post-larval) were exposed to four nominal concentrations (6.25%, 12.5%, 50% and 100% with actual polycyclic aromatic hydrocarbon (PAH) concentrations ranging from 0 to 512 µg/L) of high energy water accommodated fractions (HEWAF) under different oxic (2.0 or >5.0 mg/L) and salinity (10 or 30 ppt) regimes at 30 °C for 48 h. We found that the post-larval developmental stage was the most sensitive to oil toxicity. Median lethal concentrations during the post-larval exposures followed a treatment-dependent pattern with the highest mortality observed under hypoxic-high salinity conditions (64.55 µg/L). Real-time PCR analysis identified down regulation of target genes, encoding cytochrome P450-1α (cyp1a1), erythropoietin (epo), and the aryl hydrocarbon receptor nuclear translocator (arnt1) only when oil exposure occurred under hypoxic-high salinity conditions in treatments with PAH concentrations greater than 226 µg/L. The target genes measured in this experiment are involved in the aryl hydrocarbon receptor signaling pathway which modulates metabolism of PAHs (a major component of crude oil), and the hypoxia inducible 1-α signaling pathway which is responsible for resilience to hypoxic stress, and it is known that disruption of these pathways can lead to an array of acute and chronic effects. Our results indicated that sheepshead minnow are most sensitive to oil exposure during the post-larval developmental stage. Survival data from this age-stage also indicate that oil toxicity response is exacerbated in hypoxic and high salinity environments. The increased mortality observed during the post-larval developmental stage might be attributed to the suppression of the aryl hydrocarbon receptor signaling and the hypoxia inducible 1-α signaling pathways which is evident in by the down-regulated expression of cyp1a1, epo, and arnt1. These findings provide more information about interactions between oil and abiotic factors which enable us to make better assumptions of the ecological impacts of DWH on coastal estuaries.

Effects of the harmful algae, Alexandrium catenella and Dinophysis acuminata, on the survival, growth, and swimming activity of early life stages of forage fish.

The effects of co-occurring harmful algal blooms (HABs) on marine organisms is largely unknown. We assessed the individual and combined impacts of the toxin producing HABs, Alexandrium catenella and Dinophysis acuminata, and a non-toxin-producing HAB (Gymnodinium instriatum) on early life stages of two estuarine fish species (Menidia beryllina and Cyprinodon variegatus). Lethal (i.e. time to death) and sublethal (i.e. growth, grazing rate, and swimming activity) effects of cultured HABs were investigated for eleutheroembryo and larval life stages. Mixed algal treatments (i.e. A. catenella and D. acuminata mixtures) were often equally toxic as A. catenella monoculture treatments alone, although responses depended on the fish species and life stage. Fish exposed to toxin producing HABs died significantly sooner (i.e. <1-3 days) than controls. Significant differences in sublethal effects were also found between fed controls and toxic HAB treatments, although responses were often similar to G. instriatum or starved controls. Collectively, the results demonstrate that HABs may reduce fish productivity and fitness.

Effects of Deepwater Horizon crude oil on ocular development in two estuarine fish species, red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus).

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil have been shown to cause the dysregulation of genes important in eye development and function, as well as morphological abnormalities of the eye. However, it is not currently understood how these changes in gene expression are manifested as deficits in visual function. Embryonic red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus) were exposed to water accommodated fractions (WAFs) of weathered crude oil and assessed for visual function using an optomotor response assay in early life-stage larvae, with subsequent samples taken for histological analysis of the eyes. Larvae of both species exposed to increasing concentrations of oil exhibited a reduced optomotor response. The mean diameters of retinal layers, which play an important role in visual function and image processing, were significantly reduced in oil-exposed sheepshead larvae, though not in red drum larvae. The present study provides evidence that weathered crude oil has a significant effect on visual function in early life-stage fishes.

An embryonic staging series up to hatching for Cyprinodon variegatus: An emerging fish model for developmental, evolutionary, and ecological research.

Using multiple taxa to research development is necessary for making general conclusions about developmental patterns and mechanisms. We present a staging series for Cyprinodon variegatus as a basis for further study of the developmental biology of fishes in the genus Cyprinodon and for comparative work on teleost fishes beyond the standard models. Cyprinodon are small, euryhaline fishes, widely distributed in fresh, brackish, and hypersaline waters of southern and eastern North America. Cyprinodontids are closely related to fundulids, providing a comparative reference point to the embryological model, Fundulus heteroclitus. Ecologists and evolutionary biologists commonly study Cyprinodon, and we have been using Cyprinodon to study skull variation and its genetic basis among closely related species. We divided embryonic development of C. variegatus into 34 morphologically identifiable stages. We reference our staging series to that already defined for a related model species, Oryzias latipes (medaka) that is studied by a large community of researchers. We provide a description of the early chondrogenesis and ossification of skull and caudal fin bones during the latter stages of embryonic development. We show that Cyprinodon are tractable for studying development. Eggs can be obtained easily from breeding pairs and our study provides a staging system to facilitate future developmental studies.

The genome of Austrofundulus limnaeus offers insights into extreme vertebrate stress tolerance and embryonic development.

BACKGROUND: The annual killifish Austrofundulus limnaeus inhabits ephemeral ponds in northern Venezuela, South America, and is an emerging extremophile model for vertebrate diapause, stress tolerance, and evolution. Embryos of A. limnaeus regularly experience extended periods of desiccation and anoxia as a part of their natural history and have unique metabolic and developmental adaptations. Currently, there are limited genomic resources available for gene expression and evolutionary studies that can take advantage of A. limnaeus as a unique model system. RESULTS: We describe the first draft genome sequence of A. limnaeus. The genome was assembled de novo using a merged assembly strategy and was annotated using the NCBI Eukaryotic Annotation Pipeline. We show that the assembled genome has a high degree of completeness in genic regions that is on par with several other teleost genomes. Using RNA-seq and phylogenetic-based approaches, we identify several candidate genes that may be important for embryonic stress tolerance and post-diapause development in A. limnaeus. Several of these genes include heat shock proteins that have unique expression patterns in A. limnaeus embryos and at least one of these may be under positive selection. CONCLUSION: The A. limnaeus genome is the first South American annual killifish genome made publicly available. This genome will be a valuable resource for comparative genomics to determine the genetic and evolutionary mechanisms that support the unique biology of annual killifishes. In a broader context, this genome will be a valuable tool for exploring genome-environment interactions and their impacts on vertebrate physiology and evolution.

Embryonic and early development of the Zagros tooth-carp, Aphanius vladykovi (Actinopterygii: Cyprinodontidae).

The embryonic and early larval development of laboratory reared Zagros tooth-carp, Aphanius vladykovi Coad, 1988, are described and illustrated. Development and embryogenesis start with the external fertilization of sticky, transparent and spherical telolecithal/macrolecithal eggs with a mean diameter of 1.61± 0.12 mm and it continues with meroblastic/radial cleavage, blastulation/blastula formation, epibolic cell migration during gastrulation and organogenesis resulting in a newly hatched larvae of 5.23 ± 0.09 mm in length with attached yolk sac at about 164 hr (at 24 ± 1°C) after fertilization.

Embryonic development of the annual killifish Austrofundulus limnaeus: An emerging model for ecological and evolutionary developmental biology research and instruction.

BACKGROUND: Austrofundulus limnaeus is an annual killifish from the Maracaibo basin of Venezuela. Annual killifishes are unique among vertebrates in their ability to enter into a state of dormancy at up to three distinct developmental stages termed diapause I, II, and III. These embryos are tolerant of a wide variety of environmental stresses and develop relatively slowly compared with nonannual fishes. RESULTS: These traits make them an excellent model for research on interactions between the genome and the environment during development, and an excellent choice for developmental biology laboratories. Furthermore, A. limnaeus is relatively easy to maintain in a laboratory setting and has a high fecundity, making it an excellent candidate as an emerging model for studies of development, and for defining the limits of developmental buffering in vertebrates. CONCLUSIONS: This study reports for the first time on the detailed development of A. limnaeus and provides a photographic and illustrated atlas of embryos on the two developmental trajectories possible in this species. Developmental Dynamics 246:779-801, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Hit pause: Developmental arrest in annual killifishes and their close relatives.

Killifishes survive and persist in extreme environments by exploiting both aquatic and terrestrial habitats for egg deposition, and by adjusting the length of development to match availability of water to support larval growth and maturation. Annual killifishes persist in ephemeral bodies of water through the production of drought-tolerant embryos. Survival of the environmental stresses associated with their highly variable and seasonal habitat is supported by their ability to enter into at least two states of metabolic and developmental dormancy, diapause or quiescence. There are three stages of diapause in annual killifishes, one occurring prior to gastrulation, one about midway through development, and one in late pre-hatching embryos. Quiescence may occur at any developmental stage. In addition, delayed hatching is known to occur in close relatives of the annual killifishes, and may be superficially confused with pre-hatching diapause. These types of developmental delay are induced by different cues and serve different purposes in the life history of the species. Thus, it is likely that the molecular mechanisms that induce dormancy and support survival are unique in each case. It is imperative that we properly define these forms of developmental dormancy in our studies in order to put our results into the proper ecological and evolutionary context. Here the unique characteristics of these distinct categories of developmental delay are reviewed. Developmental Dynamics 246:858-866, 2017. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Hypoxia and Anoxia Tolerance in the Annual Killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus are routinely exposed to oxygen limitation during development and are extremely tolerant of anoxia. Importantly, tolerance of anoxia is not strictly associated with entrance into metabolic dormancy associated with diapause II, but rather any embryo will respond to anoxia by entering into a state of anoxia-induced quiescence. Hypoxia causes a reduction in the rate of development, reduced heart rates, and reduced capacities for metabolic enzyme activity in both aerobic and anaerobic pathways. Embryos of A. limnaeus begin life as oxyconformers, and transition into oxyregulators near the completion of embryonic development. As this transition occurs, extreme anoxia tolerance is lost. The rate of early development is independent of oxygen partial pressure, despite the fact that the embryos are oxyconformers. This suggests a contribution from anaerobic pathways to support early development. However, the specific pathways supporting this metabolism are unknown. The response of A. limnaeus embryos to hypoxia and anoxia is unique compared to other fishes and most other vertebrates, and thus future studies on this species may lend insight into novel mechanisms that support survival during prolonged oxygen limitation.

Developmentally arrested Austrofundulus limnaeus embryos have changes in post-translational modifications of histone H3.

Although vertebrate embryogenesis is typically a continuous and dynamic process, some embryos have evolved mechanisms to developmentally arrest. The embryos of Austrofundulus limnaeus, a killifish that resides in ephemeral ponds, routinely enter diapause II (DII), a reversible developmental arrest promoted by endogenous cues rather than environmental stress. DII, which starts at 24-26 days post-fertilization and can persist for months, is characterized by a significant decline in heart rate and an arrest of development and differentiation. Thus, A. limnaeus is a unique model to study epigenetic features associated with embryonic arrest. To investigate chromosome structures associated with mitosis or gene expression, we examined the post-translational modifications of histone H3 (phosphorylation of serine 10, mono-, di- and tri-methylation of lysine 4 or 27) in preDII, DII and postDII embryos. As seen by microscopy analysis, DII embryos have a significant decrease in the H3S10P marker for mitotic nuclei and an inner nuclear membrane localization of the H3K27me2 marker associated with silencing of gene expression. ELISA experiments reveal that the levels of methylation at H3K4 and H3K27 are significantly different between preDII, DII and postDII embryos, indicating that there are molecular differences between embryos of different chronological age and stage of development. Furthermore, in DII embryos relative to preDII embryos, there are differences in the level of H3K27me3 and H3K4me3, which may reflect critical chromatin remodeling that occurs prior to arrest of embryogenesis. This work helps lay a foundation for chromatin analysis of vertebrate embryo diapause, an intriguing yet greatly understudied phenomenon.

Fish embryos on land: terrestrial embryo deposition lowers oxygen uptake without altering growth or survival in the amphibious fish Kryptolebias marmoratus.

Few teleost fishes incubate embryos out of water, but the oxygen-rich terrestrial environment could provide advantages for early growth and development. We tested the hypothesis that embryonic oxygen uptake is limited in aquatic environments relative to air using the self-fertilizing amphibious mangrove rivulus, Kryptolebias marmoratus, which typically inhabits hypoxic, water-filled crab burrows. We found that adult mangrove rivulus released twice as many embryos in terrestrial versus aquatic environments and that air-reared embryos had accelerated developmental rates. Surprisingly, air-reared embryos consumed 44% less oxygen and possessed larger yolk reserves, but attained the same mass, length and chorion thickness. Water-reared embryos moved their opercula ∼2.5 more times per minute compared with air-reared embryos at 7 days post-release, which probably contributed to the higher rates of oxygen uptake and yolk utilization we observed. Genetically identical air- and water-reared embryos from the same parent were raised to maturity, but the embryonic environment did not affect growth, reproduction or emersion ability in adults. Therefore, although aspects of early development were plastic, these early differences were not sustained into adulthood. Kryptolebias marmoratus embryos hatched out of water when exposed to aerial hypoxia. We conclude that exposure to a terrestrial environment reduces the energetic costs of development partly by reducing the necessity of embryonic movements to dispel stagnant boundary layers. Terrestrial incubation of young would be especially beneficial to amphibious fishes that occupy aquatic habitats of poor water quality, assuming low terrestrial predation and desiccation risks.

Extreme tolerance and developmental buffering of UV-C induced DNA damage in embryos of the annual killifish Austrofundulus limnaeus.

Free-living aquatic embryos are often at risk of exposure to ultraviolet radiation (UV-R). Successful completion of embryonic development depends on efficient removal of DNA lesions, and thus many aquatic embryos have mechanisms to reverse DNA lesions induced by UV-R. However, little is known of how embryos that are able to enter embryonic dormancy may respond to UV-R exposure and subsequent DNA damage. Embryos of the annual killifish Austrofundulus limnaeus are unique among vertebrates because their normal embryonic development includes (1) a complete dispersion of embryonic blastomeres prior to formation of the definitive embryonic axis, and (2) entry into a state of metabolic depression and developmental arrest termed diapause. Here, we show that developing and diapausing embryos of A. limnaeus have exceptional tolerance of UV-C radiation and can successfully complete embryonic development after receiving substantial doses of UV-C, especially if allowed to recover in full-spectrum light. Recovery in full-spectrum light permits efficient removal of the most common type of DNA lesion induced by UV-R: cyclobutane pyrimidine dimers. Interestingly, whole-mount embryo TUNEL assays suggest that apoptosis may not be a major contributor to cell death in embryos UV-C irradiated during dispersion/reaggregation or diapause. We also observed embryo mortality to be significantly delayed by several weeks in diapausing embryos irradiated and allowed to recover in the dark. These atypical responses to UV-R induced DNA damage may be due to the unique annual killifish life history and provide insight into DNA damage repair and recognition mechanisms during embryonic dormancy.

Bmp suppression in mangrove killifish embryos causes a split in the body axis.

Bone morphogenetic proteins (Bmp) are major players in the formation of the vertebrate body plan due to their crucial role in patterning of the dorsal-ventral (DV) axis. Despite the highly conserved nature of Bmp signalling in vertebrates, the consequences of changing this pathway can be species-specific. Here, we report that Bmp plays an important role in epiboly, yolk syncytial layer (YSL) movements, and anterior-posterior (AP) axis formation in embryos of the self-fertilizing mangrove killifish, Kryptolebias marmoratus. Stage and dose specific exposures of embryos to the Bmp inhibitor dorsomorphin (DM) produced three distinctive morphologies, with the most extreme condition creating the splitbody phenotype, characterised by an extremely short AP axis where the neural tube, somites, and notochord were bilaterally split. In addition, parts of caudal neural tissues were separated from the main body and formed cell islands in the posterior region of the embryo. This splitbody phenotype, which has not been reported in other animals, shows that modification of Bmp may lead to significantly different consequences during development in other vertebrate species.

The effects of hypoxia and temperature on metabolic aspects of embryonic development in the annual killifish Austrofundulus limnaeus.

Embryos of Austrofundulus limnaeus are exceptional in their ability to tolerate prolonged bouts of complete anoxia. Hypoxia and anoxia are a normal part of their developmental environment. Here, we exposed embryos to a range of PO2 levels at two different temperatures (25 and 30 °C) to study the combined effects of reduced oxygen and increased temperature on developmental rate, heart rate, and metabolic enzyme capacity. Hypoxia decreased overall developmental rate and caused a stage-specific decline in heart rate. However, the rate of early development prior to the onset of organogenesis is insensitive to PO2. Increased incubation temperature caused an increase in the developmental rate at high PO2s, but hindered developmental progression under severe hypoxia. Embryonic DNA content in pre-hatching embryos was positively correlated with PO2. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase capacity were all reduced in embryos developing under hypoxic conditions. Embryos of A. limnaeus are able to develop normally across a wide range of PO2s and contrary to most other vertebrates severe hypoxia is not a teratogen. Embryos of A. limnaeus do not respond to hypoxia through an increase in the capacity for enzymatic activity of the metabolic enzymes lactate dehydrogenase, citrate synthase, or phosphoenolpyruvate carboxykinase. Instead they appear to adjust whole-embryo metabolic capacity to match oxygen availability. However, decreased DNA content in hypoxia-reared embryos suggests that cellular enzymatic capacity may remain unchanged in response to hypoxia, and the reduced capacity may rather indicate reduced cell number in hypoxic embryos.

Patterns of ubiquitylation and SUMOylation associated with exposure to anoxia in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus acquire extreme tolerance to anoxia during embryonic development. These embryos can survive environmental and cellular conditions that would likely result in death in the majority of vertebrate cells, despite experiencing a massive loss of ATP. It is highly likely that the initial response to anoxia must quickly alter cellular physiology to reprogram cell signaling and metabolic pathways to support anaerobiosis. Covalent protein modifications are a mechanism that can quickly act to effect large-scale changes in protein structure and function and have been suggested by others to play a key role in mammalian ischemia tolerance. Using Western blot analysis, we explored patterns of protein ubiquitylation and SUMOylation in embryos of A. limnaeus exposed to anoxia and anoxic preconditioning. Surprisingly, we report stage-specific protein ubiquitylation patterns that suggest different mechanisms for altering protein turnover in dormant and actively developing embryos that both survive long-term anoxia. Anoxic preconditioning does not appear to alter levels of ubiquitin conjugates in a unique manner. Global SUMOylation of proteins does not change in response to anoxia, but there are stage-specific changes in SUMOylation of specific protein bands. Contrary to other systems, global changes in protein SUMOylation may not be required to support long-term tolerance to anoxia in embryos of A. limnaeus. These data lead us to conclude that embryos of A. limnaeus respond to anoxia in a unique manner compared to other vertebrate models of anoxia tolerance and may provide novel mechanisms for engineering vertebrate tissues to survive long-term anoxia.

Avoidance of apoptosis in embryonic cells of the annual killifish Austrofundulus limnaeus exposed to anoxia.

Embryos of the annual killifish Austrofunduluslimnaeus have unequalled ability among vertebrates to survive long-term anoxia. Surprisingly, these embryos can survive for months in anoxia despite a large-scale decrease in ATP levels during the initial hours of anoxic exposure. These conditions are known to trigger apoptotic cell death in mammalian cells as a result of ischemia or anoxia. Anoxia-induced induction of apoptosis was investigated in four developmental stages of A. limnaeus that differ in their tolerance of anoxia, and thus may respond to anoxia uniquely. Exposure to staurosporine was used to determine if A. limnaeus cells were competent to enter apoptosis via cues other than anoxia. Apoptotic cells were identified by TUNEL assays and by measuring caspase 3/7 activity. Exposure to 48 hr of anoxia did not induce an increase in TUNEL-positive cells and generally did not lead to an increase in caspase 3/7 activity. However, treatment of anoxic embryos with 10 µM staurosporine resulted in a significant increase in caspase 3/7 activity in both normoxic and anoxic embryos. These results suggest that apoptosis is avoided in embryos of A. limnaeus following exposure to anoxia at least in part by mechanisms that prevent the activation of caspase 3/7 activity. While this mechanism remains unknown, it may be triggered by a protein kinase that can be experimentally inhibited by staurosporine.

Desiccation resistance in embryos of the killifish, Fundulus heteroclitus: single embryo measurements.

Northern killifish, Fundulus heteroclitus macrolepidotus, spawn in estuaries at high tides. Embryos may be stranded in air at stream margins as the water recedes. These aerially incubated embryos are exposed to desiccation stress and may survive and develop normally to hatching at ∼14 days post-fertilization (dpf). We developed a technique to quantitatively measure the kinetics of water loss at various developmental stages from single embryos in controlled relative humidities (RHs). Embryos were able to tolerate short periods (2 hr) of severe desiccation and survive to hatching. Mid-stage (7 dpf) embryos showed the highest degree of desiccation tolerance compared to early-stage (2 dpf) and late-stage (14 dpf) embryos. We classified the patterns of water loss into four phases, the perivitelline space (PVS) phase, the resistance phase, the desiccation phase, and the equilibration phase. In the PVS phase, water loss was rapid at all developmental stages and all RHs (∼25% of total embryo weight). The water loss rate was slower during the resistance phase. It decreased as RH increased and length of this phase was longer in mid-stage than in early- and late-stage embryos. The water loss rate and length of the desiccation phase also depended on RH. These data support the hypothesis that low permeability embryonic compartment surface membranes retard water loss significantly and promote prolonged survival of these embryos during desiccation. We also show this mechanism cannot completely account for the survival of severely desiccated embryos (especially in 23% RH) and that there must also be complementary cellular responses.

Cell cycle arrest associated with anoxia-induced quiescence, anoxic preconditioning, and embryonic diapause in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus can enter into dormancy associated with diapause and anoxia-induced quiescence. Dormant embryos are composed primarily of cells arrested in the G(1)/G(0) phase of the cell cycle based on flow cytometry analysis of DNA content. In fact, most cells in developing embryos contain only a diploid complement of DNA, with very few cells found in the S, G(2), or M phases of the cell cycle. Diapause II embryos appear to be in a G(0)-like state with low levels of cyclin D1 and p53. However, the active form of pAKT is high during diapause II. Exposure to anoxia causes an increase in cyclin D1 and p53 expression in diapause II embryos, suggesting a possible re-entry into the cell cycle. Post-diapause II embryos exposed to anoxia or anoxic preconditioning have stable levels of cyclin D1 and stable or reduced levels of p53. The amount of pAKT is severely reduced in 12 dpd embryos exposed to anoxia or anoxic preconditioning. This study is the first to evaluate cell cycle control in embryos of A. limnaeus during embryonic diapause and in response to anoxia and builds a foundation for future research on the role of cell cycle arrest in supporting vertebrate dormancy.

Gamma irradiation-induced oxidative stress and developmental impairment in the hermaphroditic fish, Kryptolebias marmoratus embryo.

This study investigated the effects of gamma radiation on the early developmental stages in hermaphroditic fish embryos of Kryptolebias marmoratus. The authors measured reactive oxygen species (ROS) level and antioxidant enzyme activities with the endpoint hatching rate after gamma irradiation of different embryonic stages. Then, the transcriptional changes of antioxidant enzyme-coding genes were evaluated by quantitative real-time reverse transcription polymerase chain reaction in response to gamma radiation on embryonic stages. Gamma radiation inhibited hatching rate and caused developmental impairment in a dose-dependent manner. Embryos showed tolerances in a developmental stage-dependent manner, indicating that early embryonic stages were more sensitive to the negative effects of gamma radiation than were later stages. After 5 Gy rate of radiation, the ROS level increased significantly at embryonic stages 2, 3, and 4 with a significant induction of all antioxidant enzyme activities. The expressions of glutathione S-transferase isoforms, catalase, superoxide dismutase (Mn-SOD, Cu/Zn-SOD), glutathione reductase, and glutathione peroxidase mRNA were upregulated in a dose-and-developmental stage-dependent manner. This finding indicates that gamma radiation can induce oxidative stress and subsequently modulates the expression of antioxidant enzyme-coding genes as one of the defense mechanisms. Interestingly, embryonic stage 1 exposed to gamma radiation showed a decreased expression in most antioxidant enzyme-coding genes, suggesting that this is also related to a lower hatching rate and developmental impairment. The results of this study provide a better understanding of the molecular mode of action of gamma radiation in aquatic organisms.

Cell cycle regulation during development and dormancy in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus can enter into a state of metabolic dormancy, termed diapause, as a normal part of their development. In addition, these embryos can also survive for prolonged sojourns in the complete absence of oxygen. Dormant embryos support their metabolism using anaerobic metabolic pathways, regardless of oxygen availability. Dormancy in diapause is associated with high ATP and a positive cellular energy status, while anoxia causes a severe reduction in ATP content and large reductions in adenylate energy charge and ATP/ADP ratios. Most cells are arrested in the G 1/G 0 phase of the cell cycle during diapause and in response to oxygen deprivation. In this paper, we review what is known about the physiological and biochemical mechanisms that support metabolic dormancy in this species. We also highlight the great potential that this model holds for identifying novel therapies for human diseases such as heart attack, stroke and cancer.