Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus

The growth, toxicity, and associated bacterial flora of 10 clonal cultures of the toxic benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus isolated from the coastal waters of southwest Puerto Rico have been examined. Clonal cultures of O. lenticularis grew more rapidly and at broader temperature ranges than those of G. toxicus. All five Ostreopsis clones were toxic, while only one of the five Gambierdiscus clones was poisonous. The degree of toxicity among poisonous clones was highly variable. The number of associated bacterial genera and their frequency of occurrence were quite variable among clones of both dinoflagellate genera. Bacterial isolates represented six genera (Nocardia, Pseudomonas, Vibrio, Aeromonas, Flavobacterium, and Moraxella) in addition to coryneform bacteria. Extracts of dinoflagellate-associated bacteria grown in pure culture were not toxic. Gambierdiscus clones were characterized by the frequent presence of Pseudomonas spp. (four of five clones) and the absence of coryneforms. In O. lenticularis, only one of five clones showed the presence of Pseudomonas spp., and Moraxella sp. was absent altogether. Detailed analyses of toxicity and associated microflora in a selected Ostreopsis clone, repeatedly cultivated (four times) over a period of 160 days, showed that peak cell toxicities developed in the late static and early negative culture growth phases. Peak Ostreopsis cell toxicities in the stationary phase of culture growth were correlated with significant increases in the percent total bacteria directly associated with these cells. Changes in the quantity of bacteria directly associated with microalgal cell surfaces and extracellular matrices during culture growth may be related to variability and degree of toxicity in these laboratory-cultured benthic dinoflagellates.     

CD4+ lipid bilayers. A model for human immunodeficiency virus type 1 coat protein binding.

gp120, the coat glycoprotein of the human immunodeficiency virus type 1 (HIV1) binds to a molecule on the surface of a class of T-lymphocytes, CD4, which is also the receptor for major histocompatibility complex class II (MHCII). To study the events that follow the interaction of gp120 with CD4, we have incorporated CD4 into lipid bilayers and recorded the electrical changes which occur after the addition of gp120. Interaction of gp120 to CD4-containing bilayers induces multistate ion-permeable channels with a maximum conductance of 380-400 picosiemens. When CD4+ bilayers were preexposed to either MHCII or to OKT4A antibody, no channels were formed after the addition of gp120. These results indicate that CD(4+)-containing bilayers bind gp120, MHCII, and OKT4A, that binding of gp120 produces ion-permeable channels, and that CD4+ bilayers can be used to assay for gp120 in the solution bathing the bilayer.     

Mutations in many genes affect aggressive behavior in Drosophila melanogaster

Background  

Aggressive behavior in animals is important for survival and reproduction. Identifying the underlying genes and environmental contexts that affect aggressive behavior is important for understanding the evolutionary forces that maintain variation for aggressive behavior in natural populations, and to develop therapeutic interventions to modulate extreme levels of aggressive behavior in humans. While the role of neurotransmitters and a few other molecules in mediating and modulating levels of aggression is well established, it is likely that many additional genetic pathways remain undiscovered. Drosophila melanogaster has recently been established as an excellent model organism for studying the genetic basis of aggressive behavior. Here, we present the results of a screen of 170 Drosophila P-element insertional mutations for quantitative differences in aggressive behavior from their co-isogenic control line.     

Bacteria Associated with Toxic Clonal Cultures of the Dinoflagellate <Emphasis Type="Italic">Ostreopsis lenticularis</Emphasis>

The marine toxic dinoflagellate Ostreopsis lenticularis has been implicated as the major vector in ciguatera seafood poisoning on the southwest coast of Puerto Rico. Studies have demonstrated that associated bacteria play a role in the ciguatoxin production and that different clonal cultures of O. lenticularis harbor different culturable bacteria. In this study, more than 125 associated bacteria from two toxic clonal cultures of O. lenticularis (no. 302 and no. 303) were analyzed utilizing polymerase chain reaction amplification of the partial small subunit ribosomal DNA (rRNA), denaturing gradient gel electrophoresis, and DNA sequencing. Approximately 50% of total bacteria identified in both cultures were a single species belonging to the Cytophaga-Flavobacter-Bacteroides complex. This bacterium was also found in six new O. lenticularis clonal cultures established 10 years after the original cultures used in this study and absent from a clonal culture of a different dinoflagellate species. The data presented here indicate a persistent and apparently specific association of this bacterium with O. lenticularis, which makes it a candidate involved in ciguatoxin production.     

Effects of palytoxin on cation occlusion and phosphorylation of the (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-ATPase

Palytoxin (PTX) inhibits the (Na(+) + K+)-driven pump and simultaneously opens channels that are equally permeable to Na+ and K+ in red cells and other cell membranes. In an effort to understand the mechanism by which PTX induces these fluxes, we have studied the effects of PTX on: 1) K+ and Na+ occlusion by the pump protein; 2) phosphorylation and dephosphorylation of the enzyme when a phosphoenzyme is formed from ATP and from P(i); and 3) p-nitro phenyl phosphatase (p-NPPase) activity associated with the (Na+, K+)-ATPase. We have found that palytoxin 1) increases the rate of deocclusion of K+(Rb+) in a time- and concentration-dependent manner, whereas Na+ occluded in the presence of oligomycin is unaffected by the toxin; 2) makes phosphorylation from P(i) insensitive to K+, and 3) stimulates the p-NPPase activity. The results are consistent with the notion that PTX produces a conformation of the Na+, K(+)-pump that resembles the one observed when ATP is bound to its low-affinity binding site. Further, they suggest that the channels that are formed by PTX might arise as a consequence of a perturbation in the ATPase structure, leading to the loss of control of the outside "gate" of the enzyme and hence to an uncoupling of the ion transport from the catalytic function of the ATPase.     

Degradation of Starch–Poly(β-Hydroxybutyrate-Co-β-Hydroxyvalerate) Bioplastic in Tropical Coastal Waters

Extruded bioplastic was prepared from cornstarch or poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) or blends of cornstarch and PHBV. The blended formulations contained 30 or 50% starch in the presence or absence of polyethylene oxide (PEO), which enhances adherence of starch granules to PHBV. Degradation of these formulations was monitored for 1 year at four stations in coastal water southwest of Puerto Rico. Two stations were within a mangrove stand. The other two were offshore; one of these stations was on a shallow shoulder of a reef, and the other was at a location in deeper water. Microbial enumeration at the four stations revealed considerable flux in the populations over the course of the year. However, in general, the overall population densities were 1 order of magnitude less at the deeper-water station than at the other stations. Starch degraders were 10- to 50-fold more prevalent than PHBV degraders at all of the stations. Accordingly, degradation of the bioplastic, as determined by weight loss and deterioration of tensile properties, correlated with the amount of starch present (100% starch >50% starch > 30% starch > 100% PHBV). Incorporation of PEO into blends slightly retarded the rate of degradation. The rate of loss of starch from the 100% starch samples was about 2%/day, while the rate of loss of PHBV from the 100% PHBV samples was about 0.1%/day. Biphasic weight loss was observed for the starch-PHBV blends at all of the stations. A predictive mathematical model for loss of individual polymers from a 30% starch–70% PHBV formulation was developed and experimentally validated. The model showed that PHBV degradation was delayed 50 days until more than 80% of the starch was consumed and predicted that starch and PHBV in the blend had half-lives of 19 and 158 days, respectively. Consistent with the relatively low microbial populations, bioplastic degradation at the deeper-water station exhibited an initial lag period, after which degradation rates comparable to the degradation rates at the other stations were observed. Presumably, significant biodegradation occurred only after colonization of the plastic, a parameter that was dependent on the resident microbial populations. Therefore, it can be reasonably inferred that extended degradation lags would occur in open ocean water where microbes are sparse.     

Discovery of a Lipid Synthesising Organ in the Auditory System of an Insect

Weta possess typical Ensifera ears. Each ear comprises three functional parts: two equally sized tympanal membranes, an underlying system of modified tracheal chambers, and the auditory sensory organ, the crista acustica. This organ sits within an enclosed fluid-filled channel–previously presumed to be hemolymph. The role this channel plays in insect hearing is unknown. We discovered that the fluid within the channel is not actually hemolymph, but a medium composed principally of lipid from a new class. Three-dimensional imaging of this lipid channel revealed a previously undescribed tissue structure within the channel, which we refer to as the olivarius organ. Investigations into the function of the olivarius reveal de novo lipid synthesis indicating that it is producing these lipids in situ from acetate. The auditory role of this lipid channel was investigated using Laser Doppler vibrometry of the tympanal membrane, which shows that the displacement of the membrane is significantly increased when the lipid is removed from the auditory system. Neural sensitivity of the system, however, decreased upon removal of the lipid–a surprising result considering that in a typical auditory system both the mechanical and auditory sensitivity are positively correlated. These two results coupled with 3D modelling of the auditory system lead us to hypothesize a model for weta audition, relying strongly on the presence of the lipid channel. This is the first instance of lipids being associated with an auditory system outside of the Odentocete cetaceans, demonstrating convergence for the use of lipids in hearing.