Culturable microflora of Artemia franciscana reared under laboratory conditions

Artemia is widely used as an initial food for larval and juvenile fish in aquaculture facilities around the world. However, several lines of research have strongly suggested that Artemia larvae may carry opportunistic pathogens such as Listonella anguillarum, thereby serving as a source of infection of fish. In the present study, we investigated the dynamics of the culturable microflora of Artemia reared under laboratory conditions, with the goal of understanding the risk of opportunistic infection mediated by this animal. After hatching decapsulated cysts of A. franciscana, the larvae were reared for an additional 27 days to examine, using the culture-dependent method, the culturable microflora of the rearing water and of washed Artemia. The results showed that Vibrionaceae, Flavobacteriaceae, Pseudoalteromonadaceae, Alteromonadaceae and Rhodobacteriaceae accounted for 8.3-35.8% of the rearing water isolates. In contrast, Vibrionaceae dominated in Artemia isolates, accounting for 79.2% of the flora. However, Vibrionaceae were not detected in either decapsulated or undecapsulated cysts, or in the algal concentrates used as feed, suggesting that Vibrionaceae is not indigenous to Artemia cysts and instead is derived primarily from natural seawater. These results strongly suggest that hatching and rearing live diets such as Artemia under sanitary conditions may reduce the risk of opportunistic infection

Abundance and diversity of predominant sulfate-reducing bacteria in the gut of pufferfish

Sulfate-reducing bacteria (SRB) exist in anaerobic environments, such as marine sediments, and produce hydrogen sulfide, which is toxic to marine animals. However, little is known about the ecology of SRB in the gut of fish. In the present study, we used molecular techniques to analyze the predominant SRB community in the gut of pufferfish inhabiting coastal areas of Japan. The density of the dissimilatory sulfite reductase alpha gene, dsrA, derived from SRB and total count of bacteria in guts of pufferfish was 4.4í—106 - 1.8í—107 copies/g and 3.5í—108 - 6.3í—109 cells/g, respectively, in all specimens. Clones of dsrA associated with Desulfobulbus oligotrophicus, a dominant SRB species, were detected in all 12 libraries, accounting for 57.7-94.3% of clones in each library. These results strongly suggest that SRB are indigenous bacteria in the gut of pufferfish and that hydrogen sulfide produced by SRB may be a risk factor for fish health

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

Oxygen tolerance of Cetobacterium somerae isolated from the gut of freshwater fish and their environments

Cetobacterium somerae is a predominant bacterium found in the gut of freshwater fish. However, being an anaerobic bacterium, its survival is believed to be prevented in oxygen-rich environments. Therefore, in this study, we investigated the oxygen tolerance of 83 C. somerae strains collected from the guts of freshwater fish, rearing water, and sediment of culture ponds and tanks. When placed in sterile bottles containing common carp- and goldfish-rearing water, C. somerae showed a 1-log decrease after 24 hours, suggesting that this organism faces challenges in growing in rearing water. Subsequently, we inoculated the bacterial strains onto agar plates and exposed them to air for 12 hours to measure the oxygen inhibition index (OII). The OII values ranged from 0.01 to 4.65 among different strains, indicating significant variation in oxygen tolerance within the bacterium. Furthermore, the OII values varied considerably depending on the isolation source, with sediment, rearing water, and gut samples showing increasing values in that order. This suggests that oxygen tolerance plays a substantial role in the ecological behavior of C. somerae

TTX-Bearing Planocerid Flatworm (Platyhelminthes: Acotylea) in the Ryukyu Islands, Japan

Polyclad flatworms comprise a highly diverse and cosmopolitan group of marine turbellarians. Although some species of the genera Planocera and Stylochoplana are known to be tetrodotoxin (TTX)-bearing, there are few new reports. In this study, planocerid-like flatworm specimens were found in the sea bottom off the waters around the Ryukyu Islands, Japan. The bodies were translucent with brown reticulate mottle, contained two conical tentacles with eye spots clustered at the base, and had a slightly frilled-body margin. Each specimen was subjected to TTX extraction followed by liquid chromatography with tandem mass spectrometry analysis. Mass chromatograms were found to be identical to those of the TTX standards. The TTX amounts in the two flatworm specimens were calculated to be 468 and 3634 μg. Their external morphology was found to be identical to that of Planocera heda. Phylogenetic analysis based on the sequences of the 28S rRNA gene and cytochrome-c oxidase subunit I gene also showed that both specimens clustered with the flatworms of the genus Planocera (Planocera multitentaculata and Planocera reticulata). This fact suggests that there might be other Planocera species that also possess highly concentrated TTX, contributing to the toxification of TTX-bearing organisms, including fish