Effects of scavenger enzymes for active oxygen such as superoxide dismutase (SOD) and catalase, and aquaporin-I inhibiters such as HgCl2 and tetraethylammonium (TEA) on the algicidal activity of arachidonic acid (AA) to the red-tide raphidophycean flagellate Heterosigma akashiwo, and also the effect of exposure to AA on fatty acid composition and morphology of the cells, and the incorporation of deuterium oxide (D2O) into the cells were examined in order to make clear the mechanisms which harmful/toxic flagellates were killed due to the effects of a highly unsaturated fatty acid.
Algicidal activity was not suppressed by the addition of SOD and/or catalase, but was suppressed by the additions of HgCl2 and TEA. The survival rate of H. akashiwo was increased by the addition of HgCl2 at 1×10-4–1×102 nM in the presence of a 300 ng mL-1 of AA (survival rate was 0％), and the maximum survival rate was 50–55％ at 1×10-2–3×10-1 nM. In the TEA, the algicidal activity was suppressed at 1×10-4–1×102 mM and the maximum survival rate was 60–65％ at 1×10-2–1×10-1 mM.
The morphology of H. akashiwo changed from an elliptic to a round, swelled shape with the survival rate decreasing, and the active swimming cell numbers also decreased upon exposure to AA. Furthermore incorporation of water into H.akashiwo, analyzed with D2O as a tracer, was promoted by exposure to AA. These phenomena, except the rounding of the shape, were suppressed by the addition of TEA. On the other hand, AA content in the cells significantly increased upon exposure to AA, and the increase was not affected by the addition of TEA.
These results suggest that a possible factor contributing to the algicidal activity of AA in H. akashiwo seems to be swelling that brings about the rupture of cells caused by changing the membrane structure into one that cannot withstand an increase in intracellular pressure resulting from accumulation of AA in the membrane and the acceleration of water intake by promoting the activity of aquaporin-I. Toxic effects of active oxygen derived from autoxidation of AA is not considered to be a major cause of the algicidal activity.
Various types of opening/closing plankton nets have been devised for depth-specific sampling. While electrically-operated multiple layer opening/closing nets are commonly used to collect samples from deep layers from well-equipped research vessels, messenger-operated opening/closing nets are useful for sampling from small boats at shallow depths. The latter nets were originally designed for horizontal or oblique tows, whereas a closing (non-opening) net is still often used for a vertical haul. However a closing net inevitably suffers from contamination when a net is allowed to obliquely descend due to ship drift caused by the wind or water currents. Previous messenger-operated opening/closing nets have also had disadvantages when they are lowered vertically, i.e. contamination with surface plankton due to incomplete closing of the net mouth and sampling failure due to tangling of the slackened bridle and/or choker ropes. To overcome these disadvantages, a new messenger-operated opening/closing net was designed for vertical hauls. The frame of the net consists of a base, two mouth rings (45 cm diameter) hinged to the base, and a same-sized center ring. The mouth rings are each equipped with a cylindrical-conical net (0.10 mm mesh), with the cod end being clamped to a rope suspending the weight lead, and is equipped with a flow meter. The operating lines consist of a “sampling wire” connected to the center ring, a “first closing wire” connected to one of the mouth rings, and a “second closing rope” connected to the double mouth rings. While the net is lowered, the first closing wire is kept under tension by gravity acting on the net frame and the weight lead tightly shuts the mouth rings like a closed clamshell. Upon deployment of the first messenger, the first closing wire is released, the sampling wire is brought under tension, and the mouth rings are opened with the aid of tension springs and wires for synchronous opening. The second messenger, deployed after sampling, causes the sampling wire to be released and the mouth rings to close again through the tension of the second closing rope. The new design is almost completely free from contamination because how tightly closed the mouths are while descending, decreases in sampling failures caused by tangling of the bridle and closing rope, and gives correct counts from flow meters because the meters are stopped from working while the mouths are closed. In addition, a high sampling efficiency is expected because of its bongo-net design when the mouths are opened. An easily-operated double release mechanism was also designed for this net. Previous mechanisms for opening/closing nets are deployed with latches along the lateral side. In such a design, a mechanism inclines against the towing line by a force driven on lateral latches. Especially when vertical hauling, the inclination sometimes does not make the messenger successfully hit the trigger. Latches of the present mechanism are placed at its bottom and their releases are turned by a spring-tensioned, swinging rod connected to the trigger. A square-shaped frame is employed for the trigger, by which a swivel can be set between the towing line and the mechanism. The mechanism can be used for multi-layer sampling if a wire stop is welded on the base plate. Using the present net and double release system, the author has collected depth-specific plankton samples monthly from 70 and 200 m depths from a small boat (five tons) for the last nine months without any significant troubles.
The cosmopolitan diatom Skeletonema costatum is widely considered to be one of the most important phytoplankton species because as a primary producer, it contributes to the productivity of global marine food chains and occasionally forms heavy blooms. New taxonomic methods using electron microscopy for fine morphological observations, and gene analysis of ribosomal DNA (rDNA) in 2005 and 2007 showed that “S. costatum” actually includes eight species: S. ardens, S. costatum sensu stricto (s.s.), S. dohrnii, S. grethae, S. grevillei, S. japonicum, S. marinoi and S. pseudocostatum. With the addition of these species to the three existing species, S. menzelii, S. subsalsum and S. tropicum, the genus Skeletonema comprises 11 species. The results of identification using both morphology and gene analysis agree well and reinforce the validity and applicability of the new classification methods. Building on this progress and my own findings over the last five years, I herein comment on the methods of identification and classification of the 11 species of the genus Skeletonema, introduce the eco-physiological characteristics and biogeography of the new Skeletonema species, and present results of population studies of S. marinoi using genetic methods. A noteworthy outcome of the new taxonomic methods was the resolution of 21 strains in the S. marinoi-dohrnii complex. The global distribution of S. costatum sensu lato (s.l.), with opportunistic features, is explained by the discovery that this species was comprised of eight related species expected to have different eco-physiological characteristics. New research has revealed that the global distribution pattern of S. japonicum could be explained by its temperature–growth characteristics. The high diversity of the genus Skeletonema is evident in Dokai Bay, Japan, where seven or eight species were reported, and four species were counted in a single sample. Interesting results from ongoing research suggest that the genetic structure of the S. marinoi population in Mariager Fjord, Denmark has been stable for over one hundred years. Hopefully, the taxonomy will continue to develop using other gene markers, and these taxonomic methods will be applied to different areas.
In the case of examining the performance of ballast water treatment systems, it is necessary to follow the "Guidelines for approval of ballast water management systems (G8)" by the IMO-MEPC. When determining the performance of a ballast water treatment system using plankton viability, the plankton cells should be counted within 6 hours after the treatment to verify if the number of viable plankton is reduced below the ballast water management standard, or the sample should be treated in such a way so as to ensure that a proper analysis can be performed. In order to count the number of viable phytoplankton more efficiently, a suitable method of staining plankton cells with neutral red (NR) is described in the present paper.
The phytoplankton that can be stained with the NR method include Bacillariophyceae, Chrysophyceae, Dinophyceae, Cryptophyceae, Raphidophyceae, Haptophyceae, Euglenophyceae and autotrophic micro-flagellates. This NR method can also be applied to general phytoplankton in other taxa and to various sizes of cells that appear in coastal areas. However, the pH value of the ballast water during the treatment must be above 7.0 and below 8.5, otherwise it may have an effect on the staining performance; therefore, a positive check is needed before the method is applied.