Masatoshi Matsumasa

The ultrastructure of the sternal gills forming a striking contrast with the coxal gills in a fresh-water amphipod (crustacea)

Sternomoera yezoensis has specialized sterna with 21 sternal gills in addition to six pairs of coxal gills. Despite a common high permeability to chloride ions, the epithelia of these two kinds of gills are diametrically opposed in the polarity of the cell membrane-mitochondria complex. The coxal gill epithelium (4-6 microm thick) is characterized by a well-developed AIS (apical infolding system) associated with a huge number of large mitochondria. The AIS exceeds two-thirds of the epithelial thickness and forms a highly sophisticated, subcuticular labyrinth. On the contrary, the sternal gill epithelium, an extension of the sternal epithelium proper, is extremely thick (10-15 microm) and is characterized by a very deep BIS (basolateral infolding system) associated with numerous slender mitochondria. The BIS reaches nine-tenths of the epithelial thickness and forms a giant, baso-lateral labyrinth. Shallower, less elaborate AIS and BIS without mitochondrial association originate from the opposite sides of these epithelia. Although AIS and BIS interpenetrate in the sternal gill epithelium, they never communicate. The results indicate that in addition to the coxal gills, the sterna with the sternal gills function as transporting as well as respiratory organs, though the functional difference between these two kinds of gills remains to be elucidated.

The osmoregulatory tissue around the afferent blood vessels of the coxal gills in the estuarine amphipods, Grandidierella japonica and Melita setiflagella.

By electron microscopy of the coxal gills in two species of estuarine amphipod crustaceans, Grandidierella japonica and Melita satifragella, we found a patch-like, specialized tissue area which consisted of unique cells closely resembling the salt-excreting cells in the gill of the brine shrimp and so-called chloride cells in teleost gills. These cells were characterized by an abundance of mitochondria, two kinds of extensive networks of cytoplasmic tubules, well-developed lamellar infoldings of the basal cell membrane, sparse microvillous projections of the apical border, and numerous large vacuoles with several incomplete partitions. The large (60 nm in diameter) and the small (30 nm) cytoplasmic tubular networks were found in the basal and the apical portions of the cell, respectively. The large networks, which were both directly and indirectly (through the lamellar system) continuous with the basal cell membrane, were regarded as extensions of the cell membrane. Both the outer walls and the partition walls of the vacuoles were reinforced with a parallel array of microtubules. The results suggest that this unique tissue plays an important role in the active transport of electrolytes to maintain a constant osmotic pressure of the hemolymph under widely fluctuating salinities of the estuarine environments.

“Igloo” construction by the ocypodid crab, Dotilla myctiroides (Milne-Edwards) (Crustacea; Brachyura): the role of an air chamber when burrowing in a saturated sandy substratum

Dotilla myctiroides, an inhabitant of tropical sandy shores, builds a unique burrow structure in semi-fluid, unstable sand, in addition to a vertical burrow constructed in well-drained and firm sand. The crab rotates in the sand, forming a circular wall of sand pellets around it and subsequently roofs the wall with pellets. The resulting burrow structure, termed an “igloo”, encloses a small quantity of air together with the crab itself. In this study, the function of the igloo was examined and compared to that of the vertical burrow structure. In the air-filled chamber beneath the sand surface, which results from the construction of the igloo, the crab continued to scoop up the sand from the lower surface of the chamber, and attached the scooped pellets to the ceiling of the chamber. Consequently, the crab, together with the chamber, continually moved deeper into the sand until it was below the water level. In contrast, 40% of the crabs artificially buried in sand without a surrounding air chamber remained where they had been buried 12 h later. This indicated that the air chamber greatly facilitates vertical movement of the crab. The making of the igloo is considered to be an adaptation that enables the construction of an air chamber in semi-fluid sand, where a vertical burrow cannot be constructed, because the sand is not sufficiently firm to prevent it from collapsing. The descent of the crab into the sand through the construction of an igloo may prevent an encounter with dangers such as predators arriving with the incoming tide, a role played by the construction of a vertical burrow when the sandy substratum is firm and well-drained.

Macroalgae and seagrass contribution to gastropods in sub-tropical and temperate tidal flats

Macroalgal and seagrass communities are widely distributed in marine and brackish shallow water and have high productivity. The primary production in marine costal areas is often transported to intertidal flats by waves and currents and beached on the flats. Thus, we hypothesized that the macroalgae and seagrasses are food sources for benthic communities on intertidal flats where some gastropod species often dominate. We performed comparisons of food sources among different gastropod species on sub-tropical and temperate tidal flats (26° and 38°N, respectively), and used isotope mixing models using carbon and nitrogen stable isotopes. A mixing model for stable isotopes, IsoSource, revealed that main food sources for three snails were macroalgae (50-56%) and seagrass (39-45%) at the temperate site. The contributions of terrestrial plants, sediment organic matter and benthic microalgae were weaker than those of macroalgae and seagrasses. At the sub-tropical site, snails fed mainly on macroalgae. The differences in food sources between snail species were not remarkable, although the nitrogen values were slightly different. It would thus appear that macroalgae and seagrass play an important role in the food webs not only in their own habitats but also on the adjacent tidal flats.

ULTRASTRUCTURAL EVIDENCE FOR OSMOREGULATORY FUNCTION OF THE STERNAL EPITHELIA IN SOME GAMMARIDEAN AMPHIPODS

ABSTRACT The ion-permeabilities and the ultrastructure of the sternal epithelia were studied in 7 species of gammaridean amphipods. Evidence has been obtained that the sterna can be divided into 2 categories: ion-transporting and non-ion-transporting types. The sterna of Sternomoera japonica, Grandidierella japonica, Melita setiflagella, and Corophium uenoi, belonging to the former category, are covered by ion-permeable cuticular layers lined with a thick epithelium, which is characterized by both a shallow apical infolding system (AIS) and a well-developed basolateral infolding system (BIS). The AIS is composed of frequent lamellar infoldings without mitochondrial association originating from the apical sides of the epithelia and involves virtually every part of the apical cell membrane, forming elaborate intercellular channels in the subcuticular space. In contrast, the BIS consists of deep infoldings of the basolateral cell membranes and extensive interdigitations between epithelial cells, which are both associated with a large number of mitochondria. This BIS involves the greater part of the cell membrane and constitutes a complicated, giant labyrinth of the intercellular spaces above the basal lamina. The AIS and BIS never communicate in the sternal epithelia, though they sometimes interpenetrate. On the other hand, the sterna of Jesogammarus jesoensis, Jesogammarus hinumensis, and Haustorioides sp., belonging to the latter category, are composed of an ion-impermeable, thick cuticular layer and a thin epithelium characterized by a paucity of mitochondria and complete lack of the BIS and AIS, which are common to those of the ordinary epithelium of the body surface. The results suggest a rather widespread occurrence in amphipods of a variety of extrabranchial ion-permeable areas of the body surface. They also suggest that the sterna lined with thick, specialized epithelia, together with the gills, are involved in the active transport of electrolytes in osmoregulation as well as respiration.

Practical applications of molecular biological species identification of forensically important flies.

Species identification of the early colonizers on a corpse, such as Calliphoridae and Sarcophagidae, provides useful information for estimating the postmortem interval (PMI). However, the morphology of adult flies and nymphs is very similar among species of the same genus, and for many forensic scientists accurate identification is very difficult within their routine work. We present a simple and time-saving method for identification of forensically important flies by comparing sequenced 304 bp of mitochondrial DNA cytochrome oxidase gene subunit I (mtDNA COI). Eggs and larvae were recovered from the corpse during forensic autopsies and then reared in a growth chamber under controlled conditions. The emerging adults were killed by ethyl acetate and male specimens were observed under a stereomicroscope to identify the species based on the shape of the genitalia. Subsequently, DNA was extracted from identified specimens and sequenced mtDNA COI, resulting in the identification of 8 species of 4 genera of forensically important diptera. Moreover, the molecular biological species identification was useful in forensic practice, and there were cases in which analysis of DNA extracted from puparia supposed to be early colonizers of the corpse provided useful information for estimating the PMI. We also report sequencing of mtDNA COI of the green bottle fly, Lucilia ampullacea, newly collected from a corpse in Iwate prefecture.

Specialized Ion-Transporting Epithelium around the Blood Vessel of the Coxal Gills in a Deep-Sea Amphipod, Eurythenes gryllus

ABSTRACT Histological examination showed that the deep-sea amphipod Eurythenes gryllus possesses a unique tissue area around the afferent blood vessel of the gill, which exhibits well-developed infoldings of the basolateral cell membrane with many mitochondria. The location (i.e., around the afferent blood vessel) and the above ultrastructural characteristics indicate that this unique tissue is homologous with a type of osmoregulatory tissue which was described for the patchlike area of gills in two estuarine amphipods Grandidierella japonica and Melita setiflagella. We review the presence/absence of this specialized tissue for 12 species (including the above 3 species) belonging to 8 genera collected from deep-sea, coastal, brackish, supralittoral, and fresh-water areas. All but Sternomoerajaponica, the distribution of which is limited to fresh-water seepage flows in forests, possessed the tissue. The discovery of the specialized tissue in E. gryllus confirms the widespread occurrence of the tissue in amphipod species.

Colonization of the Restored and Newly Created Tidal Flats by Benthic Animals in the Sanriku Region of Northern Japan

Tsunamis and subsidence caused by the Great East Japan Earthquake in 2011 created and restored some intertidal areas along the Sanriku coastline of northern Japan. Otomo-ura is one such area in Iwate Prefecture. The tidal flat of Otomo-ura had been reclaimed for farmland 50 years prior but has “returned to the sea” after the tsunamis. Our 3-year-long qualitative and quantitative investigation of benthic fauna after the tsunamis reveals that a variety of animals have quickly colonized the newly created habitat. In August 2012, 2013, and 2014, the number of species observed was 27, 51, and 59, respectively. Rapid colonization was also observed for another newly created habitat in the Unosumai River estuary, which is situated about 40 km north of Otomo-ura. We find that that habitat heterogeneity between the peripheral and central areas and microhabitats provided by primary (e.g., sand and cobbles) and secondary substrates (e.g., oyster’s and algal bodies) both contributed to increased species richness and population density. Although direct-developing species, which lack planktonic larval stages, had hardly been expected to colonize the restored and newly created vacant tidal flats, a direct-developing gastropod Batillaria attramentaria has been observed in Otomo-ura since August 2014. Colonization by the direct-developing gastropod and DNA analyses for 15 populations, including one from Otomo-ura, indicate that regional management of source populations will be necessary to prevent colonization by Euspira fortunei, another direct-developing alien predator, and to diminish the effects of parasitic Perkinsus on the host clam population.

Effects of the Great East Japan Earthquake on Intertidal Macrobenthos in Iwate Prefecture

To determine the effects of the Great East Japan Earthquake and resulting tsunamis on the distribution of marine benthic macrofauna throughout Iwate Prefecture, the fauna of three tidal flats was compared before and after this catastrophic event. Because this prefecture has a history of tsunami events, floodgates and seawalls were established along its coastline following the tsunami in 1933 as a disaster prevention strategy. Consequently, the intertidal environment throughout the region had been already modified before the 2011 tsunamis. Despite this former loss of habitat, a number of benthic invertebrates occurred in the tidal areas before the intertidal disturbance due to the 2011 tsunamis. More importantly, 52–76 % of taxa of benthic invertebrates previously recorded in this region were found in the tidal area after the tsunamis’ disturbance. Additionally, several taxa that are considered threatened (according to the Japanese Association of Benthology) appeared in these and newly created tsunami-flooded habitats after 2011. Our data suggest that the marine macrobenthos in this region, including these threatened taxa, were tolerant of the tsunami disturbance or comprised opportunistic taxa capable of rapidly colonizing new habitats. However, the recovery patterns of benthic invertebrates varied across sites: remarkable reduction of tidal flat decreased its biological diversity, but creation of new tidal habitats strikingly increased taxa richness. The reconstruction of coastal structures should be planned considering preservation of the remaining and newly created tidal flats. Future disaster risk management should take into consideration both the maintenance costs of these structures and the effects they have on biodiversity and ecosystem functioning in intertidal flats.

Observation of cavitation bubble cloud using a stroboscopic x-ray generator

The radiographic characteristics of the condenser-discharge stroboscopic x-ray generator and its application to high-speed radiography are described. This generator employs a condenser- discharge medical x-ray generator in conjunction with a new grid-control unit and consists of the following major components: a high-voltage control unit, a condenser unit having a Cockcroft circuit, a grid-control unit, a high- voltage grid-control unit, and a rotating-anode x-ray tube unit. The main condenser of about 500 nF in the unit is charged up to 120 kV by the circuit, and the electric charges in the condenser are discharged to the triode by the grid control unit in conjunction with a high-voltage grid-control unit. The high-voltage grid-control unit is employed in order to insulate the negative high-voltage grid and cathode electrodes from the high-speed grid control unit with almost the ground potential. In this generator the anode and cathode electrodes are applied to high-voltages with maximum values of +60 kV and -60 kV, respectively. Although the tube voltage decreased during the discharging for generating x- rays, the maximum value was equivalent to the initial charging voltage of the main condenser. The maximum values of the tube current and the repetition rate were about 0.5 A and 32 kHz, respectively. The pulse width of the x-rays ranged from 0.01 to 1.0 ms, and the maximum shot number had a value of 32. At a constant filament (cathode) temperature, the x-ray intensity increased according to increases in the charging voltage and to increases in the duration, and the maximum intensity with a duration of 1.0 ms and a charging voltage of 100 kV was 0.66 (mu) C/kg at 1.0 m from the x-ray source. The dimensions of the focal spot had values of about 1 X 1 mm, and both the inner and outer triggering for producing pulse x-rays can be employed. The high-speed radiography was primarily performed by the multiple-shot radiography using a new computed radiography (CR) system in conjunction with the computer- display system with an operation system of Windows NT Workstation 4.0.