Atsushi Ishimatsu

Effects of high CO2 seawater on the copepod (Acartia tsuensis) through all life stages and subsequent generations

We studied the effects of exposure to seawater equilibrated with CO(2)-enriched air (CO(2) 2380 ppm) from eggs to maturity and over two subsequent generations on the copepod Acartia tsuensis. Compared to the control (CO(2) 380 ppm), high CO(2) exposure through all life stages of the 1st generation copepods did not significantly affect survival, body size or developmental speed. Egg production and hatching rates were also not significantly different between the initial generation of females exposed to high CO(2) and the 1st and 2nd generation females developed from eggs to maturity in high CO(2). Thus, the copepods appear more tolerant to increased CO(2) than other marine organisms previously investigated for CO(2) tolerance (i.e., sea urchins and bivalves). However, the crucial importance of copepods in marine ecosystems requires thorough evaluation of the overall impacts of marine environmental changes predicted to occur with increased CO(2) concentrations, i.e., increased temperature, enhanced UV irradiation, and changes in the community structure and nutritional value of phytoplankton.

Hydroxyl radical generation by red tide algae.

The unicellular marine phytoplankton Chattonella marina is known to have toxic effects against various living marine organisms, especially fishes. However, details of the mechanism of the toxicity of this plankton remain obscure. Here we demonstrate the generation of superoxide and hydroxyl radicals from a red tide unicellular organism, C. marina, by using ESR spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-t-butyl-alpha-phenylnitrone (PBN), and by using the luminol-enhanced chemiluminescence response. The spin-trapping assay revealed productions of spin adduct of superoxide anion (O2-) (DMPO-OOH) and that of hydroxyl radical (.OH) (DMPO-OH) in the algal suspension, which was not observed in the ultrasonic-ruptured suspension. The addition of superoxide dismutase (500 U/ml) almost completely inhibited the formation of both DMPO-OOH and DMPO-OH, and carbon-centered radicals were generated with the disappearance of DMPO-OH after addition of 5% dimethyl sulfoxide (Me2SO) and 5% ethanol. Furthermore, the generation of methyl and methoxyl radicals, which are thought to be produced by the reaction of hydroxyl radical and Me2SO under aerobic condition, was identified using spin trapping with a combination of PBN and Me2SO. Luminol-enhanced chemiluminescence assay also supported the above observations. These results clearly indicate that C. marina generates and releases the superoxide radical followed by the production of hydroxyl radical to the surrounding environment. The velocity of superoxide generation by C. marina was about 100 times faster than that by mammalian phagocytes per cell basis. The generation of oxygen radical is suggested to be a pathogenic principle in the toxication of red tide to susceptible aquaculture fishes and may be directly correlated with the coastal pollution by red tide.

Mudskippers store air in their burrows

Mudskipper fishes can maintain their metabolism while they are confined in mudflat burrows filled with oxygen-depleted water, and their eggs, deposited in the burrows, can develop under severely hypoxic conditions. How they cope with such conditions has been unclear. We report here that a mudskipper species Periophthalmodon schlosseri (Fig. 1) accumulates air in its burrows. This behaviour seems to be an adaptation to provide oxygen for burrow-dwelling fish and for embryos developing in the burrows.

Oxygen-radical-mediated toxic effects of the red tide flagellate Chattonella marina on Vibrio alginolyticus

Toxic mechanisms of the red tide flagellate, Chattonella marina, collected in 1985 from Kagoshima Bay, Japan, were studied at the subcellular level. C. marina was found to reduce ferricytochrome c at a rate related to the concentration of plankton cells. Ca. 50% of the cytochrome c reduction was inhibited by the addition of 100 U superoxide dismutase ml-1. These results suggest that a part of the cytochrome c reduction was caused by a superoxide anion which was extracellulary released from C. marina. Moreover, a small amount of hydrogen peroxide was detected in the C. marina suspension using the fluorescence spectrophotometric assay method. The identity of the hydrogen peroxide was confirmed by its reaction with 500 U catalase ml-1. It is thus proposed that C. marina produces harmful active oxygen radicals and therefore exhibits a toxic effect on surrounding living organisms. In agreement with these results, C. marina strongly inhibited the proliferation of marine bacteria, Vibrio alginolyticus, in a plankton/bacteria co-culture system. The growth inhibition of bacteria caused by C. marina was related to the density and the metabolic potential of C. marina. Ruptured plankton showed no toxic effect on the bacteria. Furthermore, the toxic effect of C. marina on V. alginolyticus was completely suppressed by the addition of catalase and superoxide dismutase. In addition to these radical-scavenging enzymes, a chemical scavenger, sodium benzoate, also had a protective effect. These results suggest that oxygen radicals are important in the toxic action of C. marina.

Will krill fare well under Southern Ocean acidification

Antarctic krill embryos and larvae were experimentally exposed to 380 (control), 1000 and 2000 µatm pCO2 in order to assess the possible impact of ocean acidification on early development of krill. No significant effects were detected on embryonic development or larval behaviour at 1000 µatm pCO2; however, at 2000 µatm pCO2 development was disrupted before gastrulation in 90 per cent of embryos, and no larvae hatched successfully. Our model projections demonstrated that Southern Ocean sea water pCO2 could rise up to 1400 µatm in krills depth range under the IPCC IS92a scenario by the year 2100 (atmospheric pCO2 788 µatm). These results point out the urgent need for understanding the pCO2-response relationship for krill developmental and later stages, in order to predict the possible fate of this key species in the Southern Ocean.

Mechanism of superoxide anion generation in the toxic red tide phytoplankton Chattonella marina: possible involvement of NAD(P)H oxidase

Red tide phytoplankton Chattonella marina is known to produce reactive oxygen species (ROS), such as superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radical (&z.rad;OH), under normal physiological conditions. Although several lines of evidence suggest that ROS are involved in the mortality of fish exposed to C. marina, the mechanism of ROS generation in C. marina remains to be clarified. In this study, we found that the cell-free supernatant prepared from C. marina cells showed NAD(P)H-dependent O(2)(-) generation, and this response was inhibited by diphenyleneiodonium, an inhibitor of mammalian NADPH oxidase. When the cell-free supernatant of C. marina was analyzed by immunoblotting using antibody raised against the human neutrophil cytochrome b558 large subunit (gp91phox), a main band of approximately 110 kDa was detected. The cell surface localization of the epitope recognized with this antibody was also demonstrated in C. marina by indirect immunofluorescence. Furthermore, Southern blot analysis performed on genomic DNA of C. marina with a probe covering the C-terminal region of gp91phox suggested the presence of a single-copy gene coding for gp91phox homologous protein in C. marina. These results provide evidence for the involvement of an enzymatic system analogous to the neutrophil NADPH oxidase as a source of O(2)(-) production in C. marina.

Mudskippers brood their eggs in air but submerge them for hatching.

SUMMARY Intertidal mudflats are highly productive ecosystems that impose severe environmental challenges on their occupants due to tidal oscillations and extreme shifts in habitat conditions. Reproduction on mudflats requires protection of developing eggs from thermal and salinity extremes, O2 shortage, dislodgement by currents, siltation and predation. Mudskippers are air-breathing, amphibious fishes, and one of few vertebrates that reside on mudflats. They lay their eggs in mud burrows containing extremely hypoxic water, raising the question of how the eggs survive. We found that the Japanese mudskipper Periophthalmus modestus deposits its eggs on the walls of an air-filled chamber within its burrow. To ensure adequate O2 for egg development, the burrow-guarding male mudskipper deposits mouthfuls of fresh air into the egg chamber during each low tide, a behaviour that can be upregulated by egg-chamber hypoxia. When egg development is complete the male, on a nocturnal rising tide, removes the egg-chamber air and releases it outside the burrow. This floods the egg chamber and induces egg hatching. Thus, P. modestus has developed a reproductive strategy that allows it to nurture eggs in this severe habitat rather than migrating away from the mudflat. This requires that mudskipper eggs be specialized to develop in air and that the air-breathing capacity of the egg-guarding male be integrated in a complex behavioural repertoire that includes egg guarding, ferrying air to and from the egg chamber, and sensing O2 levels therein, all in concert with the tidal cycle.

Inhibitory effect of the iron chelator Desferrioxamine (Desferal) on the generation of activated oxygen species by Chattonella marina

Recent studies demonstrated that the toxic red tide phytoplankton Chattonella spp. produce activated oxygen species such as superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH), which may be responsible for the toxicity of this flagellate. However, the mechanism behind the production of these oxygen radicals and H2O2 by Chattonella spp. is largely unknown, and the physiological significance of activated oxygen species for Chattonella spp. is also unclear. In the present study, we investigated the involvement of iron in the generation of O2-and H2O2 by C. marina. The generation of O2-by C. marina was related to the growth phase; the highest rate of O2-production was observed during the exponential growth phase. However, no such increase during the exponential growth phase was observed in C. marina growing in an iron-deficient medium, even though the growth of C. marina was not significantly affected by iron-deficiency during the first 4 d. In addition, the iron chelator desferrioxamine (Desferal) strongly inhibited the generation of both O2-and H2O2 by C. marina in a concentration-dependent manner. The growth of C. marina was also inhibited by Desferal. Furthermore, in the presence of 500 μM Desferal, C. marina-induced growth inhibition of the marine bacteria Vibrio alginolyticus was almost completely abolished. These results suggest that iron is required for the generation of activated oxygen species by C. marina, as well as for its own growth.

Aerial ventilatory responses of the mudskipper, Periophthalmodon schlosseri, to altered aerial and aquatic respiratory gas concentrations.

Periophthalmodon schlosseri is a mudskipper which uses the vascularized buccopharyngeal cavity as a respiratory organ. The fish construct mud burrows that contain hypoxic water, but store air inside the burrows. Because the burrow gas is frequently hypoxic and hypercapnic, the effects of altered respiratory gas concentrations on the aerial ventilation frequency (V(F)), inspiratory tidal volume (V(T)) and minute volume (V(M)=V(F)xV(T)) of P. schlosseri were studied by pneumotachography. Both total buccopharyngeal gas volume (V(BP)) and V(T) scaled significantly with body mass (mass exponents=1.10 and 1.03, respectively), and V(T)/V(BP) was 0.54+/-0. 05 (S.E.M., n=6). V(BP), expressed as a percentage of body volume, was much higher (16%) than in other air-breathing gobies (2-4%). When fish respired in normoxic air and water, V(F) was 0.25+/-0.04 breaths min(-1), V(T) 7.6+/-0.6 ml 100 g(-1), and V(M) 1.80+/-0.18 ml 100 g(-1) min(-1). Aquatic hypoxia did not significantly affect V(F), V(T), or V(M). In both moderate (P(O(2))=10 kPa) and severe (P(O(2))=5 kPa) aerial hypoxia, V(F) and V(M) increased significantly. V(T) increased significantly only during severe aerial hypoxia. In aerial hypercapnia, V(F) and V(M) increased significantly.

Air breathing of aquatic burrow-dwelling eel goby, Odontamblyopus lacepedii (Gobiidae: Amblyopinae)

SUMMARY Odontamblyopus lacepedii is an eel goby that inhabits both coastal waters and intertidal zones in East Asia, including Japan. The fish excavates burrows in mudflats but, unlike the sympatric amphibious mudskippers, it does not emerge but stays in the burrows filled with hypoxic water during low tide. Endoscopic observations of the field burrows demonstrated that the fish breathed air in the burrow opening; air breathing commenced 1.3 h following burrow emersion, when water PO2 was ∼2.8 kPa, with an air-breathing frequency (fAB) of 7.3±2.9 breaths h–1 (mean ± s.d., N=5). Laboratory experiments revealed that the fish is a facultative air breather. It never breathed air in normoxic water (PO2=20.7 kPa) but started bimodal respiration when water PO2 was reduced to 1.0–3.1 kPa. The fish held air inside the mouth and probably used the gills as gas-exchange surfaces since no rich vascularization occurred in the mouth linings. As is known for other air-breathing fishes, fAB increased with decreasing water PO2. Both buccal gas volume (VB) and inspired volume (VI) were significantly correlated with body mass (Mb). At a given Mb, VI was nearly always equal to VB, implying almost complete buccal gas renewal in every breathing cycle. A temporal reduction in expired volume (VE) was probably due to a low aerial gas exchange ratio (CO2 elimination/O2 uptake). Air breathing appears to have evolved in O. lacepedii as an adaptation to aquatic hypoxia in the burrows. The acquisition of the novel respiratory capacity enables this species to stay in the burrows during low tide and extends the resident time in the mudflat, thereby increasing its chances of tapping the rich resources of the area.