Oxidative stress-mediated deleterious effects of hypoxia in the brackish water flea Diaphanosoma celebensis.

In this study, we investigated the acute toxicity, in vivo effects, oxidative stress, and gene expression changes caused by hypoxia on the brackish water flea Diaphanosoma celebensis. The no-observed-effect concentration (NOEC) of 48 h of hypoxia exposure was found to be 2 mg/L O2. Chronic exposure to NOEC caused a significant decline in lifespan but had no effect on total fecundity. The induction of reactive oxygen species increased in a time-dependent manner over 48 h, whereas the content of antioxidant enzymes (superoxide dismutase and catalase) decreased. The transcription and translation levels were modulated by hypoxia exposure. In particular, a significant increase in hemoglobin level was followed by up-regulation of hypoxia-inducible factor 1α gene expression and activation of the mitogen-activated protein kinase pathway. In conclusion, our findings provide a better understanding of the molecular mechanism of the adverse effects of hypoxia in brackish water zooplankton.

Distribution of pufferfish saxitoxin- and tetrodotoxin-binding protein homolog and tetrodotoxin in the brain and pituitary of juvenile tiger puffer Takifugu rubripes.

Pufferfish saxitoxin- and tetrodotoxin (TTX)-binding protein (PSTBP) is considered to transfer TTX between tissues. The immunohistochemical distribution of PSTBP-homolog (PSTBPh) and TTX in the brain and pituitary of hatchery-reared juvenile tiger puffer Takifugu rubripes was investigated. PSTBPh was observed mainly in the pars intermedia of the pituitary. TTX was only detected in a TTX-fed fish in the neurohypophysis of the pituitary and in several other brain regions. The relationship between PSTBPh and TTX is discussed.

Toxicity and speciation of inorganic arsenics and their adverse effects on in vivo endpoints and oxidative stress in the marine medaka Oryzias melastigma.

Here, we investigate the effects of acute and chronic exposure to arsenate (AsV) and arsenite (AsIII) in the marine medaka Oryzias melastigma. In vivo effects, biotransformation, and oxidative stress were studied in marine medaka exposed to the two inorganic arsenics for 4 or 28 days. An investigation of embryonic development revealed no effect on in vivo parameters, but the hatching rate increased in the group exposed to AsIII. Exposure to AsIII also caused the greatest accumulation of arsenic in medaka. For acute exposure, the ratio of AsV to AsIII was higher than that of chronic exposure, indicating that bioaccumulation of inorganic arsenic can induce oxidative stress. The largest increase in oxidative stress was observed following acute exposure to AsIII, but no significant degree of oxidative stress was induced by chronic exposure. During acute exposure to AsV, the increase in the enzymatic activity of glutathione-S-transferase (GST) was twice as high compared with exposure to AsIII, suggesting that GST plays an important role in the initial detoxification process. In addition, an RNA-seq-based ingenuity pathway analysis revealed that acute exposure to AsIII may be related to cell-cycle progression. A network analysis using differentially expressed genes also revealed a potential link between the generation of inflammatory cytokines and oxidative stress due to arsenic exposure.

Neuronal responses of melanin-concentrating hormone and corticotropin-releasing hormone to background color in the self-fertilizing fish, Kryptolebias marmoratus.

We examined neuronal responses of hypothalamic melanin-concentrating hormone (MCH) and corticotropin-releasing hormone (CRH) to background color in the self-fertilizing fish, Kryptolebias marmoratus. Fish were individually reared in lidless white or black cylindrical plastic containers for 15 days. The number of MCH-immunoreactive (ir) cell bodies in the nucleus lateralis tuberis (NLT) of the hypothalamus was significantly greater in the white-acclimated fish, while no significant differences were observed in the nucleus anterior tuberis (NAT) of the hypothalamus. Significant differences were not seen in the number of CRH-ir cell bodies in the NLT between the groups. The body of the white- and black-acclimated fish appeared lighter and darker, respectively, compared with the baseline color. In the black-acclimated fish, feeding activity was significantly greater with a tendency toward higher specific growth rate compared with the observations in white-acclimated fish. No significant inter-group cortisol level differences were observed. These results indicate that background color affects MCH neuronal activity in the NLT as well as body color adaptation but does not affect CRH neuronal activity in K. marmoratus.

The brain of the wild toxic marine pufferfishes accumulates tetrodotoxin.

We have previously detected tetrodotoxin (TTX) in the brain of the wild toxic torafugu Takifugu rubripes by immunohistochemistry and LC/MS analysis. We have also indicated that TTX is a stress-relieving substance in the brain and reduces agonistic interactions in torafugu juveniles. Although the toxicity of marine pufferfish in the Japanese waters has been extensively examined for food hygiene, whether wild toxic pufferfish generally possess TTX in the brain has not been investigated. In the present study, we examined the presence of TTX in the brain of several wild toxic marine pufferfishes such as kusafugu T. alboplumbeus, komonfugu T. flavipterus, shosaifugu T. snyderi, okinawafugu Chelonodontops patoca, and in wild non-toxic pufferfishes such as shirosabafugu Lagocephalus spadiceus and yoritofugu Sphoeroides pachygaster. We also examined tsumugihaze Yongeichthys criniger, known to possess TTX in the skin, viscera, and gonad. TTX was extracted from the brain, liver, skin, and muscle and was analyzed by LC/MS. TTX was detected in the brain as well as in the liver, skin, and muscle in kusafugu, komonfugu, shosaifugu, okinawafugu, and tsumugihaze. In shirosabafugu, low level of TTX (0.8 mouse unit/g-brain) was detected in the brain in 1 out of 3 individuals. In yoritofugu, no TTX was detected in any of the tissues. We conclude that the brain is also an organ that contains TTX in the wild toxic marine pufferfishes.

Iron reproductive toxicity of marine rotifer sibling species: Adaptation to temperate and tropical habitats.

Iron (Fe), a trace metal in coastal waters has increased significantly due to anthropogenic activities, however, few studies have examined its toxicity to marine organism reproduction and associated mechanisms. We employed two marine rotifers, the temperate Brachionus plicatilis, and tropical B. rotundiformis to investigate the toxicity of iron (FeSO4•7H2O) and its deleterious effects on reproductive features in females (sexual fecundity, abnormal resting eggs, and swimming speed) and males (lifespan, swimming speed, and spermatozoa quality) under lethal and sub-lethal exposure. The 24 h median lethal concentration (LC50) of iron was determined as 0.9 and 1.7 µg/mL per ng of dry weight for B. plicatilis and B. rotundiformis, respectively. During sub-lethal iron (20-75 µg/mL) exposure, higher iron (≥ 20 µg/mL for B. plicatilis and ≥ 45 µg/mL for B. rotundiformis) induced rotifer sexual toxicity especially in normal resting egg development and production. These were supported by the data of male shorter lifespan, poor sperm vitality, and rotifer behavioral changes as the iron concentration increased. Iron effects on swimming behavior, slower males and faster females, should reduce male/female encounter rates associated with inactive fertilized egg (resting egg) production. Two rotifer species exhibited different iron-response patterns in genetic and enzymatic activities including iron homeostasis-maintaining related Fe-S protein, and oxidative/antioxidant related lipid peroxidation product (MDA), superoxidase dismutase/SOD, catalase/CAT, and cytochrome P450 under acute iron exposure. Antioxidant activities were vulnerable in B. plicatilis but kept activities in B. rotundiformis, which may attribute to their temperate and tropical habitat adaptations.

Effects of crowding stress on the hypothalamo-pituitary-interrenal axis of the self-fertilizing fish, Kryptolebias marmoratus.

We tested whether crowding stress affects the hypothalamo-pituitary-interrenal (HPI) axis of the self-fertilizing fish, Kryptolebias marmoratus, which is known to be aggressive in the laboratory conditions but sometimes found as a group from a single land crab burrow in the wild. The projection of corticotropin-releasing hormone (CRH) neurons to the adrenocorticotropic hormone (ACTH) cells in the pituitary was confirmed by dual-label immunohistochemistry; CRH-immunoreactive (ir) fibers originating from cell bodies located in the lateral tuberal nucleus (NLT) of the hypothalamus were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Then, fish were reared solitary or in pairs for 14 days, and the number of CRH-ir cell bodies in the NLT of the hypothalamus and cortisol levels in the body without head region were compared. The number of CRH-ir cell bodies and cortisol levels were significantly higher in paired fish. These results indicate that crowding stress affects the HPI axis in K. marmoratus which thrive in small burrows with limited water volume.

Species-specific effects of iron on temperate and tropical marine rotifers in reproduction, lipid and ROS metabolisms.

Two euryhaline rotifers, the temperate species Brachionus plicatilis and tropical species Brachionus rotundiformis, were used to investigate the effects of iron (FeSO4·7H2O), an essential trace metal, on reproductive patterns and lifetables, including the metabolism of lipid and reactive oxygen species (ROS). B. plicatilis was more sensitive to iron with regard to sexual reproduction. While iron had no significant effect on the population growth at 0-48 µg/mL, it caused a decrease in the resting egg production. B. plicatilis exposed to 6 and 12 µg/mL of iron showed an increase in the intracellular ROS levels and a decrease in the neutral lipid content in sexual organs, accompanied by downregulation of antioxidant components CuZnSOD and two cytochromes (CYP clan 2&3). These patterns suggested that iron-induced oxidative stress was not neutralized by its antioxidant defense system, thus negatively affecting the fecundity of fertilized mictic females. However, B. rotundiformis showed a dose-dependent increase in population growth with extended lifespan and positive sexual reproduction in response to 0-24 µg/mL iron. Furthermore, compared to Fe-exposed B. plicatilis, B. rotundiformis showed better antioxidant mechanism, whereas genes involved in lipid synthesis (citrate lyase, mitochondrial CYP) and reproduction (vasa, sirtuin-2) were significantly upregulated compared to the control, implying that B. rotundiformis was likely to have higher resilience in response to iron-induced oxidative stress. These findings suggest that iron is likely to cause interspecific interactions in the B. plicatilis species complex, whereas the tropical species B. rotundiformis may have evolved an effective defense mechanism against iron-induced stress.

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.

Author Correction: Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The reference genome of the selfing fish Kryptolebias hermaphroditus: Identification of phases I and II detoxification genes.

The selfing fish Kryptolebias hermaphroditus has unique reproductive system for self-fertilization, making genetically homozygous offsprings. Here, we report on high density genetic map-based genome assembly for the K. hermaphroditus Panama line (PanRS). The numbers of scaffolds were 5212 and the genome was 683,992,224 bp (N50 = 27.45 Mb). The length of anchored scaffold onto 24 linkage groups was 652,231,070 bp (95.3% of genome) with 0.01% of the gap and 39.33% of GC content and complete Benchmarking Universal Single-Copy Orthologs value was 96.6%. The numbers of annotated genes were 36,756 (average gene length 1368 bp) with the GC content of 54.1%. To examine the difference between the two sister species in the genus Kryptolebias, we compared the genomes of K. hermaphroditus PanRS and Kryptolebias marmoratus PAN line on the composition of transposable elements. To demonstrate applications of genome library, phase I and II detoxification related gene families have been analyzed, and compared the syntenies containing loci of CYP and GST genes on linkage groups. This K. hermaphroditus genome information will be helpful for a better understanding on genome-wide mechanistic view of detoxification and antioxidant-related genes over evolution in the view of fish environmental ecotoxicology.

Tetrodotoxin functions as a stress relieving substance in juvenile tiger puffer Takifugu rubripes.

We tested whether tetrodotoxin (TTX) functions as a stress relieving substance in puffer fish. We orally administered TTX to the juveniles of hatchery-reared non-toxic tiger puffer Takifugu rubripes and measured the effects of TTX on brain corticotropin-releasing hormone (CRH) mRNA expression and plasma cortisol levels in comparison with effects in non-toxic juveniles. Firstly, the reciprocal connections of CRH and adrenocorticotropic hormone (ACTH) were confirmed by dual-label immunohistochemistry. CRH-immunoreactive (ir) cell bodies were detected in the hypothalamus and CRH-ir fibers were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Next, a TTX-containing diet (2.35 mouse units (517 ng)/g diet) or a non-toxic diet were fed to the fish for 28 days under a recirculating system. Standard length and body weight became significantly larger in the TTX-treated group. The degree of loss of the caudal fin, which is an indicator of the degree of agonistic interactions, where high values show a higher loss of caudal fin of a fish due to nipping by other individuals, was significantly lower in the TTX-treated group. Relative CRH mRNA expression levels in the brain and cortisol levels in the plasma were significantly lower in the TTX-treated group. These results indicate that TTX functions as a stress relieving substance by affecting the CRH-ACTH-cortisol axis and reducing agonistic interactions in tiger puffer juveniles.

Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis).

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species.

Localization of three forms of gonadotropin-releasing hormone in the brain and pituitary of the self-fertilizing fish, Kryptolebias marmoratus.

The localization of gonadotropin-releasing hormone (GnRH) in the brain and pituitary of the self-fertilizing mangrove killifish Kryptolebias marmoratus was examined by immunohistochemistry and in situ hybridization to understand its neuroendocrine system. The genome assembly of K. marmoratus did not have any sequence encoding GnRH1, but sequences encoding GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH) were found. Therefore, GnRH1 was identified by in silico cloning. The deduced amino acid sequence of the K. marmoratus GnRH1 (mature peptide) was identical to that of the medaka GnRH. GnRH1 neurons were detected in the ventral part of the preoptic nucleus by immunohistochemistry and in situ hybridization, and GnRH1-immunoreactive (ir) fibers were observed throughout the brain. GnRH1-ir fibers were in close contact with luteinizing hormone (LH)-ir cells in the pituitary using double immunohistochemistry. GnRH2 neurons were detected in the midbrain tegmentum by immunohistochemistry and in situ hybridization. Although GnRH2-ir fibers were observed throughout the brain, they were not detected in the pituitary. GnRH3 neurons were detected in the lateral part of the ventral telencephalic area by both methods. GnRH3-ir fibers were observed throughout the brain, and a few GnRH3-ir fibers were in close contact with LH-ir cells in the pituitary. These results indicate that GnRH1 and possibly GnRH3 are responsible for gonadal maturation through LH secretion and that all three forms of GnRH function as neurotransmitters or neuromodulators in the brain of K. marmoratus.

Complete mitochondrial genome of the monogonont rotifer Brachionus rotundiformis (Rotifera, Brachionidae).

The two complete mitochondrial genomes were sequenced from the monogonont rotifer Brachionus rotundiformis. The genome sequences were 10,268 bp and 11,703 bp in size, and the gene order and contents were identical with those of B. koreanus but were different in tRNA-Cys with B. plicatilis mitochondrial genomes. Of the 12 protein-coding genes (PCGs), five genes (ND1, ATP6, ND5, CO3, ND3) had incomplete stop codons. Furthermore, the start codon of ND4 and CO3 gene was ATA, while the start codon of other PCGs was ATG. The base composition of B. rotundiformis mitogenome shows an anti-G bias (12.05% and 10.24%) on the second and third position of the PCGs, respectively.

A Genetic Map for the Only Self-Fertilizing Vertebrate.

The mangrove killifish Kryptolebias marmoratus, and its close relative Kryptolebias hermaphroditus, are the only vertebrate species known to reproduce by self-fertilization due to functional ovotestis development. To improve our understanding of their genomes, we constructed a genetic map. First, a single F1 fish was made by artificial fertilization between K. marmoratus and K. hermaphroditus strains. F2 progeny were then obtained by self-fertilization of the F1 fish. We used RAD-seq to query genomic DNAs from the two parental strains, the F1 individual and 49 F2 progeny. Results identified 9904 polymorphic RAD-tags (DNA markers) that mapped to 24 linkage groups, corresponding to the haploid chromosome number of these species. The total length of the map was 1248 cM, indicating that about one recombination occurred for each of the 24 homologous chromosome pairs in each meiosis. Markers were not evenly distributed along the chromosomes: in all chromosomes, many markers (> 8% of the total markers for each chromosome) mapped to chromosome tips. Centromeres suppress recombination, and this uneven distribution is probably due to the species' acrocentric chromosomes. Mapped marker sequences were compared to genomic sequences of medaka and platyfish, the next most closely related species with sequenced genomes that are anchored to genetic maps. Results showed that each mangrove killifish chromosome corresponds to a single chromosome of both platyfish and medaka, suggesting strong conservation of chromosomes over 100 million years of evolution. Our genetic map provides a framework for the K. marmoratus/K. hermaphroditus genome sequence and an important resource for understanding the biology of hermaphroditism.

Correlation between microbiota and growth in Mangrove Killifish (Kryptolebias marmoratus) and Atlantic cod (Gadus morhua).

The vertebrate gut is host to large communities of bacteria, and one of the beneficial contributions of this commensal gut microbiota is the increased nutritional gain from feed components that the host cannot degrade on its own. Fish larvae of similar age and under the same rearing conditions often diverge with regards to growth. The underlying reasons for this could be differences in genetic background, feeding behavior or digestive capacity. Both feeding behavior and digestion can be influenced by differences in the microbiota. To investigate possible correlations between the size of fish larvae and their gut microbiota, we analyzed the microbiota small and large genetically homogenous killifish and genetically heterogeneous cod larvae by Bray-Curtis Similarity measures of 16S DNA DGGE patterns. A significant difference in richness (p = 0.037) was observed in the gut microbiota of small and large killifish, but the overall gut microbiota was not found to be significantly different (p = 0.13), indicating strong genetic host selection on microbiota composition at the time of sampling. The microbiota of small and large cod larvae was significantly different with regards to evenness and diversity (p = 0.0001), and a strong correlation between microbiota and growth was observed.

Complete mitochondrial genome of the mangrove killifish Kryptolebias hermaphroditus (Cyprinodontiformes, Rivulidae).

The complete mitochondrial genome was sequenced from the mangrove killifish (Kryptolebias hermaphroditus). The genome sequence was 17,487 bp in size, and the gene order and contents were identical with those of the congeneric species (K. marmoratus) in the genus Kryptolebias with emphasis on the second control region (795 bp). Of 13 protein-coding genes (PGCs), 5 genes (ND2, CO2, CO3, ND3, and Cytb) had incomplete stop codons as shown in K. marmoratus. Furthermore, the stop codon of ND6 gene was AGG, while the start codon of CO1 gene was GTG. The base composition of K. hermaphroditus mitogenome showed an anti-G bias (13.45% and 8.19%) on the second and third position of the protein-coding genes (PCGs), respectively.

Larval pufferfish protected by maternal tetrodotoxin.

Marine pufferfish contain tetrodotoxin (TTX), an extremely potent neurotoxin. All species of the genus Takifugu accumulate TTX in the liver and ovaries, although the tissue(s) in which it is localized can differ among species. TTX is the major defense strategy the pufferfish appears to use against predators. TTX is also used as a male-attracting pheromone during spawning. Here we demonstrate an additional (and unexpected) use of maternal TTX in the early larval stages of the Takifugu pufferfish. Predation experiments demonstrated that juveniles of all the species of fish used as predators ingested pufferfish larvae, but spat them out promptly. Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) analysis revealed that the pufferfish larvae contain a small quantity of TTX, which is not enough to be lethal to the predators. Immunohistochemical analysis with anti-TTX monoclonal antibody revealed that the TTX is primarily localized in the body surface of the larvae as a layer of protection. Our study showed the female parent of the Takifugu pufferfish vertically transfers TTX to the larvae through its accumulation in the ovaries, and subsequent localization on the body surface of the larvae.

Complete mitochondrial genome of the monogonont rotifer, Brachionus koreanus (Rotifera, Brachionidae).

The complete mitochondrial genome was obtained from the assembled genome data sequenced by next generation sequencing (NGS) technology from the monogonont rotifer Brachionus koreanus. The mitochondrial genome of B. koreanus was composed of two circular chromosomes designated as mtDNA-I (10,421 bp) and mtDNA-II (11,923 bp). The gene contents of B. koreanus were identical with previously reported B. plicatilis mitochondrial genomes. However, gene orders of B. koreanus showed one rearrangement between the two species. Of 12 protein-coding genes (PCGs), 3 genes (ATP6, ND1, and ND3) had an incomplete stop codon. The A + T base composition of B. koreanus mitochondrial genome was high (68.81%). They also showed anti-G bias (12.03% and 10.97%) on the second and third position of PCGs as well as slight anti-C bias (15.96% and 14.31%) on the first and third position of PCGs.