18S rRNA gene sequences of leptocephalus gut contents, particulate organic matter, and biological oceanographic conditions in the western North Pacific.

Eel larvae apparently feed on marine snow, but many aspects of their feeding ecology remain unknown. The eukaryotic 18S rRNA gene sequence compositions in the gut contents of four taxa of anguilliform eel larvae were compared with the sequence compositions of vertically sampled seawater particulate organic matter (POM) in the oligotrophic western North Pacific Ocean. Both gut contents and POM were mainly composed of dinoflagellates as well as other phytoplankton (cryptophytes and diatoms) and zooplankton (ciliophoran and copepod) sequences. Gut contents also contained cryptophyte and ciliophoran genera and a few other taxa. Dinoflagellates (family Gymnodiniaceae) may be an important food source and these phytoplankton were predominant in gut contents and POM as evidenced by DNA analysis and phytoplankton cell counting. The compositions of the gut contents were not specific to the species of eel larvae or the different sampling areas, and they were most similar to POM at the chlorophyll maximum in the upper part of the thermocline (mean depth: 112 m). Our results are consistent with eel larvae feeding on marine snow at a low trophic level, and feeding may frequently occur in the chlorophyll maximum in the western North Pacific.

Dynamics of seagrass bed microbial communities in artificial Chattonella blooms: A laboratory microcosm study.

The influence of algicidal and growth-inhibiting bacteria in a seagrass (Zostera marina) bed, and their capability of controlling blooms of the fish-killing raphidophyte flagellate, Chattonella antiqua, were examined in laboratory microcosm experiments. Bacterial communities in seawater collected from the seagrass bed and Z. marina biofilm suppressed artificial Chattonella blooms in the presence of their natural competitors and predators. Phylogenetic analysis suggest that considerable numbers of bacteria that suppress Chattonella, including algicidal or growth-inhibiting bacteria isolated from seagrass biofilm and seawater from the seagrass bed, are members of Proteobacteria that can decompose lignocellulosic compounds. A direct comparison of partial 16S rRNA gene sequences (500 bp) revealed that the growth-limiting bacterium (strain ZM101) isolated from Z. marina biofilm belonged to the genus Phaeobacter (Alphaproteobacteria) showed 100% similarity with strains of growth-limiting bacteria isolated from seawater of both the seagrass bed and nearshore region, suggesting that the origin of these growth-limiting bacteria are the seagrass biofilm or seawater surrounding the seagrass bed. This study demonstrates that Chattonella growth-limiting bacteria living on seagrass biofilm and in the adjacent seawater can suppress Chattonella blooms, suggesting the possibility of Chattonella bloom prevention through restoration, protection, or introduction of seagrass in coastal areas.

Molecular diet analysis of Anguilliformes leptocephalus larvae collected in the western North Pacific.

Natural diets of leptocephalus larvae have been enigmatic. In this study, we collected DNA samples from the gut contents and body surface of leptocephali belonging to the five Anguilliform families (Anguillidae, Chlopsidae, Congridae, Muraenidae, and Serrivomeridae) from the northwest Pacific and performed next-generation 18S rDNA sequencing. Wide variety of eukaryotes was detected in both samples, from which eight eukaryotic groups (jellyfish, conoid parasite, tunicate, copepod, krill, segmented worm, fungi, and dinoflagellate) were selected on the basis of abundance. All groups except conoid parasites were common in both the samples. Cnidarian 18S rDNA reads were the most abundant in both the samples; however, the number of samples having cnidarian reads and the read counts were significantly higher in the body surface scraping samples than in the gut content samples, regardless of careful rinsing of the body surface. These results indicate that the cnidarian DNAs are most likely found because of cross contamination from the body surface and/or environment. 18S rDNA read counts of copepod and tunicate in the gut contents were greater than or comparable with those in the body surface scraping samples, which may correspond to the previous observations of fecal pellets and larvacean houses in the leptocephali gut. Thus, the present study supports previous implications that leptocephali utilize detritus materials, so called marine snow.

Algicidal and growth-inhibiting bacteria associated with seagrass and macroalgae beds in Puget Sound, WA, USA.

The algicidal and growth-inhibiting bacteria associated with seagrasses and macroalgae were characterized during the summer of 2012 and 2013 throughout Puget Sound, WA, USA. In 2012, Heterosigma akashiwo-killing bacteria were observed in concentrations of 2.8×106CFUg-1 wet in the outer organic layer (biofilm) on the common eelgrass (Zostera marina) in north Padilla Bay. Bacteria that inhibited the growth of Alexandrium tamarense were detected within the biofilm formed on the eelgrass canopy at Dumas Bay and North Bay at densities of ∼108CFUg-1 wet weight. Additionally, up to 4100CFUmL-1 of algicidal and growth-inhibiting bacteria affecting both A. tamarense and H. akashiwo were detected in seawater adjacent to seven different eelgrass beds. In 2013, H. akashiwo-killing bacteria were found on Z. marina and Ulva lactuca with the highest densities of ∼108CFUg-1 wet weight at Shallow Bay, Sucia Island. Bacteria that inhibited the growth of H. akashiwo and A. tamarense were also detected on Z. marina and Z. japonica at central Padilla Bay. Heterosigma akashiwo cysts were detected at a concentration of 3400cystsg-1 wet weight in the sediment from Westcott Bay (northern San Juan Island), a location where eelgrass disappeared in 2002. These findings provide new insights on the ecology of algicidal and growth-inhibiting bacteria, and suggest that seagrass and macroalgae provide an environment that may influence the abundance of harmful algae in this region. This work highlights the importance of protection and restoration of native seagrasses and macroalgae in nearshore environments, in particular those regions where shellfish restoration initiatives are in place to satisfy a growing demand for seafood.

Down-regulation of EGFR prolonged cell growth of glioma but did not increase the sensitivity to temozolomide.

BACKGROUND: Malignant glioma is an invasive disease of the central nervous system. One of the factors that regulate growth of these tumors is expression of epidermal growth factor receptor (EGFR) in the cells. This study investigated the effects of down-regulation of EGFR on cell proliferation, cell cycle and cytotoxicity to antineoplastic agent. MATERIALS AND METHODS: A short hairpin RNA transcription vector targeting EGFR was transfected into KNS42 cells. Growth curve, cell cycle and sensitivity to temozolomide of the cells were assessed. RESULTS: Transfection inhibited EGFR expression by 50.5%. It prolonged cell doubling time by 25.7%. However, it did not meaningfully alter the cell cycle populations nor increase sensitivity to temozolomide. CONCLUSION: Suppressing expression of EGFR inhibited cell proliferation. However, unlike PTEN expression or ROCK1 down-regulation, it did not alter the cell cycle or increase sensitivity to temozolomide.

The effect of PTEN on proliferation and drug-, and radiosensitivity in malignant glioma cells.

BACKGROUND: Deletions or mutations of the phosphatase and tensin homolog (PTEN) are frequently observed in malignant glioma and are responsible for progression of the disease. Since the molecule is a promising target for gene therapy, the effects of PTEN on glioma proliferation in combination with the anti-neoplastic agent, temozolomide, and ionizing radiation were investigated. MATERIALS AND METHODS: An adenoviral vector encoding PTEN was used. After infection, changes in proliferation, the cell cycle, as well as drug- and radiosensitivity were investigated. RESULTS: Expression of PTEN led to a 1.21-fold prolongation of the doubling time of the cells. It reduced G(1) and increased G(2)/M populations. Forced PTEN expression conferred sensitivity to temozolomide and/or ionizing radiation. CONCLUSION: In addition to counteracting cell proliferation, expression of PTEN presented advantages in the chemo- and radiosensitivity of glioma cells. Methods for up-regulation of PTEN may have a role in increasing the efficacy of current adjuvant therapies.

Effect of inhibition of the ROCK isoform on RT2 malignant glioma cells.

BACKGROUND: Malignant glioma is one of the most intractable diseases in the human body. Rho-kinase (ROCK) is overexpressed and has been proposed as the main cause for the refractoriness of the disease. Since efficacious treatment is required, this study investigated the effect of inhibition of ROCK isoforms. MATERIALS AND METHODS: The short hairpin RNA transcription vector was transfected into the RT2 rat glioma cell line and the characteristics of the cells were investigated. The effect of nimustine hydrochloride (ACNU) anti-neoplastic agent on cells was also measured. RESULTS: Inhibition of ROCK isoforms did not alter cell growth. Cell cycle analysis revealed that ROCK1 down-regulation reduced the G(0) phase population and ROCK2 down-regulation reduced the G(2)/M phase population. When ROCK1-down-regulated cells were exposed to ACNU, they demonstrated susceptibility to the agent. CONCLUSION: The roles of ROCK1 and ROCK2 may be different in glioma cells. Furthermore, the combination of ROCK1 down-regulation and an anti-neoplastic agent may be useful for the therapy of malignant glioma.