Egg viability decreases rapidly with time since ovulation in the rainbow darter Etheostoma caeruleum: implications for the costs of choosiness.

Egg viability in the rainbow darter Etheostoma caeruleum, a fish apparently lacking female mate choice, was found to decline rapidly after ovulation. It was observed that the majority of a female's clutch may fail to hatch if she is prevented from mating for as little as 6 h. These data suggest that exercising female mate preferences may be selectively disfavoured in E. caeruleum due to the high cost of delaying mating.

Influence of sex and habitat on the size and shape of anal and dorsal fins of the blackstripe topminnow Fundulus notatus.

The size and shape of the anal and dorsal fin in the blackstripe topminnow Fundulus notatus from lake and stream habitats across multiple ages and sexes were examined. Differences in the size and shape of anal and dorsal fins were sex-specific and not related to habitat differences. Males have longer and more pointed anal fins and longer, larger and more pointed dorsal fins than females. These sex differences occur predominantly in the older age class. The angle (i.e. pointedness) of the dorsal and anal fins is tightly correlated suggesting that fins follow a similar growth trajectory as individuals become sexually mature.

Reproductive isolation between two darter species is enhanced and asymmetric in sympatry.

Robust reproductive isolation was found between the rainbow darter Etheostoma caeruleum and the orangethroat darter Etheostoma spectabile, as more offspring were produced when conspecific males and females were crossed as compared with heterospecific crosses. Furthermore, fewer eggs resulted from heterospecific crosses involving sympatric E. spectabile females than those using allopatric E. spectabile females, while a similar pattern was not observed in heterospecific crosses using E. caeruleum females. These results suggest that reinforcement, i.e. selection for pre-zygotic reproductive barriers driven by reduced hybrid fitness, may have contributed to the evolution and maintenance of reproductive barriers between these potentially hybridizing species in sympatry.

The effects of water depth and light on oviposition and egg cannibalism in the bluefin killifish Lucania goodei.

This study showed that sex and depth had strong effects on egg cannibalism, whereas water clarity (clear v. tea-stained) had no effect on cannibalism or oviposition in the bluefin killifish Lucania goodei. These results are consistent with the extreme levels of iteroparity in L. goodei where females appear to spread their eggs across multiple locations and depths presumably to avoid egg predation.

Carotenoid biosynthesis in Rhodospirillum rubrum: effect of pteridine inhibitor.

A known inhibitor of pteridine utilization (4-phenoxy,2,6-diamino pyridine) blocks the synthesis of colored carotenoids in the photosynthetic bacterium Rhodospirillum rubrum. In many ways the effect is similar to the inhibition of the synthesis of colored carotenoids by diphenylamine. This inhibition is probably independent of other effects of pteridine on photosynthetic electron transport since it is not as readily reversible as the total inhibition of photosynthetic activity by pteridine analogs.

Quantitative determination of intracellular depolymerase activity in Pseudomonas oleovorans inclusions containing poly-3-hydroxyalkanoates with long alkyl substituents.

Research regarding the accurate, quantitative degradation of novel poly-3-hydroxyalkanoates has been restricted by the absence of an appropriate monitoring technique. The calibration of a gas chromatograph to poly-3-hydroxyoctanoate reveals a linear relationship between the area under gas chromatograph tracings and polymer weight. With this new method, poly-3-hydroxy-octanoate granules isolated from Pseudomonas oleovorans, which were incubated at 30 degrees C in an alkaline buffer, exhibited a linear degradation rate. Degradation was inhibited by the presence of Triton X-100 and phenylmethylsulfonyl fluoride. The depolymerase was demonstrated to be associated with the polymer granule complex and most likely possessed serine residues at its active site.

Protein organization on the PHA inclusion cytoplasmic boundary.

Polyhydroxyalkanoate (PHA) cellular inclusions consist of polyesters, phospholipids, and proteins. Both the polymerase and the depolymerase enzymes are active components of the structure. Recently, proteins associated with these inclusions have been described in a number of bacterial species. In order to further clarify the structure and function of these proteins in relation to polymer inclusions, ultrastructural studies of isolated polymer inclusions were initiated. The surface boundary characteristics of polymer inclusions, produced by several genera of bacteria, two different Pseudomonas putida deletion mutants and by Escherichia coli recombinants, were examined. The recombinant E. coli carried either the PHB biosynthesis operon (phaCAB) from Ralstonia eutropha alone, or both this operon and a gene encoding an inclusion surface protein of R. eutropha (phaP). The results support two suggestions: (i) specific genes in the PHA gene cluster code for the proteins forming the surface boundary arrays which characterize the polymer inclusion; and (ii) transfer of such a gene would result in subcellular compartmentalization of accumulating polymer. Although the proteins appear to serve a similar function among different genera, nevertheless, the different surface proteins are encoded by a variety of non-homologous genetic sequences.

Investigation of the function of proteins associated to polyhydroxyalkanoate inclusions in Pseudomonas putida BMO1.

Polyhydroxyalkanoate (PHA) granule associated proteins from Pseudomonas oleovorans were purified and the N-terminal sequences of two major proteins migrating in sodium dodecyl sulfate polyacrylamide gels with a relative molecular mass of 18 and 43 kDa (GA1 and GA2, respectively) were analyzed. Radiolabeled degenerate probes deduced from these amino acid sequences were used to identify genomic DNA fragments from P. oleovorans and Pseudomonas putida encoding GA1 and GA2. DNA sequence analysis of the fragments obtained from P. putida revealed that the genes encoding these proteins were adjacent to phaC2 and ORF3, the PHA synthase II gene and an open reading frame of unknown function, respectively, found at the P. oleovorans and P. aeruginosa PHA synthase gene locus. The open reading frames encoding GA1, GA2 and ORF3 or smaller fragments beginning at GA1 were inactivated by chromosomal insertion of the Tn5 kanamycin resistance gene block (neo). When these mutants were grown on mineral salts agar media under nitrogen limitation, containing gluconate or decanoate as carbon sources, they appeared more translucent than the wild-type grown under similar conditions. Gas-chromatographic analysis of the cellular dry mass revealed that the mutant strains accumulated 30-50% less PHA than the P. putida wild type.

Microbial inclusions with special reference to PHA inclusions and intracellular boundary envelopes.

Polyhydroxyalkanoates (PHAs), in the form of metabolic storage reserves, are assembled in intracellular cytoplasmic inclusions, often called granules. This review discusses both the structure and function of this assembly. In addition an overview of other microbial cellular inclusions is presented. This is not a compilation of all such structures but a description of those that are similar in many ways to either the structure or function of the PHA inclusions and are made up of monolayer envelopes and their storage compounds. Not unique, such inclusions provide many similar examples which, in turn, provide useful analogies to the PHA inclusions. A study of the PHA inclusions has been carried out in a comparative electron microscope examination and by protein analysis of a number of organisms and E. coli transformants.

Production of unsaturated polyesters by Pseudomonas oleovorans.

Pseudomonas oleovorans was grown separately on 3-hydroxy-6-octenoic acid and 3-hydroxy-7-octenoic acid as the only carbon source and under ammonium nutrient-limiting conditions to produce storage polyesters. The polyesters produced contained mainly unsaturated C8 units. Small amounts of both the saturated and the unsaturated C6 units were also present, but only about 1% of the saturated 3-hydroxyoctanoate units was detected. The polyester obtained from 3-hydroxy-6-octenoic acid, which was a mixture of the cis and trans isomers, also contained units with cis and trans double bonds. The weight average molecular weights of the polymers produced were in the range of 339,000-383,000 as determined by g.p.c. relative to polystyrene, with Mw/Mn ratios of 1.8-2.1. The mechanism of PHA formation from n-octene previously reported is discussed in relation to the present results, and the two were found to be in good agreement.

Bacterial polyesters containing branched poly(beta-hydroxyalkanoate) units.

Pseudomonas oleovorans was grown on mixtures of methyloctanoates with n-octanoate. Polymers were also obtained from organisms grown on pure 7-methyloctanoate, but not from pure 5- or 6-methyloctanoate. The polyesters obtained from 7-methyloctanoate and from its mixtures with n-octanoate contained units with the methyl branches in the pendant group, as did the copolymers from the mixtures of 5- and 6-methyloctanoate with n-octanoate. The methyl branched repeating units contained two diastereomers, and the 13C-n.m.r. spectra of these polymers indicated that the 5-methyloctanoate units had a higher content of one of the two isomers, but not in the 6-methyloctanoate units. The weight average molecular weights of the copolyesters produced were in the range of 220,000 to 410,000, with Mw/Mn ratios of 1.7 to 1.9.

Sequential production of two different polyesters in the inclusion bodies of Pseudomonas oleovorans.

When Pseudomonas oleovorans was grown on a mixture of 5-phenylvaleric acid, PVA, and nonanoic acid, NA, the reserve polyester produced included both a homopolymer and a copolymer. The homopolymer poly-3-hydroxy-5-phenylvalerate, PHPV, contained only 3-hydroxy-5-phenylvalerate units, while the copolymer contained the same long chain 3-hydroxyalkanoates as those present in the copolymer poly-3-hydroxynonanoate, PHN, which is produced from acid alone. The intracellular location of each of these polymers was determined by selective staining of the inclusion body granules with ruthenium tetraoxide and examination by transmission electron microscopy showed that both types of polyesters occurred in the same granule. PHN was present in the center of the granule, while PHPV accumulated around the PHN in the inclusion body. The proteins associated with the inclusion bodies were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In all cases, two different polymerase enzymes of molecular weight 59 and 55 KDa were present, indicating that the same polymerase enzyme system was responsible for the production of both PHN and PHPV. Attempts were made to produce a random copolymer containing both alkyl and phenylalkyl repeat units by varying the growth conditions, but a mixture of PHN and PHPV was always produced instead.

Intracellular depolymerase activity in isolated inclusion bodies containing polyhydroxyalkanoates with long alkyl and functional substituents in the side chain.

The in vitro degradation of isolated Pseudomonas oleovorans inclusion bodies containing either poly-3-hydroxynonanoate (PHN), or poly(-3-hydroxy-5-phenylvalerate) (PHPV), or a mixture of these two polymers was investigated. When incubated at 30 degrees C and pH 9, inclusion bodies containing either polyhydroxyoctanoate (PHO), PHN or PHPV exhibited similar degradation rates of approximately 0.94 (+/- 3%) mg/h. The PHN and PHPV components for inclusion bodies containing a mixture of PHN and PHPV showed similar degradation rates; that is the ratios showed little change and remained at approximately 50 wt.% (+/- 3%) for each component. These results contrast markedly with in vivo studies for similar inclusion bodies in whole cells. The results suggest that the synthesis and degradation of these novel polyhydroxyalkanoates by P. oleovorans proceeds by the same enzymatic pathway. In addition, comparisons between the in vivo and in vitro polymer degradation suggest that the activity of the intracellular depolymerase does not control the rate limiting step of PHPV degradation in vivo. Instead, the presence of an aromatic group in the repeating units of this polymer may inhibit the utilization of the monomeric units of PHPV as a reserve carbon source by the cells.

Ability of the phototrophic bacterium Rhodospirillum rubrum to produce various poly (beta-hydroxyalkanoates): potential sources for biodegradable polyesters.

Studies have been carried out in order to optimize growth and culture conditions for the intracellular formation of poly(beta-hydroxyalkanoates) (PHA) in the phototrophic, purple, non-sulphur bacterium Rhodospirilum rubrum. Its potential to produce novel copolymers was investigated. Recently, it has become of industrial interest to evaluate these polyesters as potentially biodegradable plastics for a wide range of possible applications. On an industrial scale, the use of photosynthetic bacteria could harness sunlight as an energy source for the production of these materials. R. rubrum was grown anaerobically in the light on different linear and branched beta-hydroxycarboxylic acids and various n-alkanoic acids. Under nitrogen-limiting conditions a PHA content of up to 45% of cellular dry weight was detected. When R. rubrum was grown on different concentrations of various n-alkanoic acids, intracellular PHA production was detected on all acids used. In most of the cases, the storage polymer contained beta-hydroxybutyrate (HB) and beta-hydroxyvalerate (HV) monomer units. Grown on n-alkanoic acids with a chain length of four carbon atoms and more, R. rubrum produced a copolymer containing the beta-hydroxyhexanoate (HC) repeating unit in addition to the HB and HV monomer. Using beta-hydroxyheptanoic acid as the carbon source, a polyester which contained HB, HV, HC, and beta-hydroxyheptanoate was formed. These copolyesters represent a novel class of biodegradable thermoplastics. The results demonstrate the metabolic flexibility of R. rubrum to form many different types of polyesters which might substitute plastics synthesized from petrochemicals.

Intracellular depolymerase functionality and location in Pseudomonas oleovorans inclusions containing polyhydroxyoctanoate.

Microbial poly-3-hydroxyoctanoate inclusion bodies produced by Pseudomonas oleovorans when grown on n-octanoic acid, are complex macromolecular structures consisting of polyester, organized paracrystalline lattice arrays and lipids. While it is known that the polymer in the granules maintains its native, amorphous state while it is surrounded by the components of this complex, the precise functions of the various components during polymer production and utilization have yet to be established. By utilizing electron microscopy, SDS-PAGE, and gel filtration chromatography along with in vitro assays for depolymerase activity, the present study demonstrates that a protein species with molecular weight of approximately 32 kDa is the depolymerase protein of the polymer inclusion. When exogenous carbon was exhausted, cell viability required utilization of the stored polyester. Under these conditions, the concentration of the depolymerase increased while the concentrations of the polymerase decreased. Thus, the association of the depolymerase with the granules was shown to be under metabolic regulation relative to the polymerase. The results from the present studies show that careful manipulation of the substrate concentration can selectively, and differentially, alter the level of inclusion associated proteins as well as the quantity and quality of the polyester which is accumulated.

Intracellular depolymerase and polyhydroxyoctanoate granule integrity in Pseudomonas oleovorans.

When polyhydroxyoctanoate (PHO) was produced by Pseudomonas oleovorans during a regimen of intermittent feeding on octanoic acid, there was a significant change in both the polymer associated proteins and the composition of the enclosed polymer. The polymer granules were isolated with their protein coat intact and the enzymatic hydrolysis of the polymer within this cell free system was determined. The degradation rate for the PHO in these native granules reached a maximum of 1.17 mg/h at an optimum pH of 9 when incubated at 30 degrees C. A study of the effect of various inhibitors on depolymerase activity suggested that the enzyme most likely has disulfide linkages and serine residues at its active site. Ultrastructure studies suggested this loss of enzyme activity was correlated with significant organizational degeneration in the proteins associated with the PHO inclusion body. Once solubilized from the granule, the depolymerase itself remained enzymatically active, and addition of this released material to other granule preparations increased the rate of polymer granule degradation. Similarly, when colloidal suspensions of purified, amorphous PHO were placed in contact with that depolymerase, they also underwent rapid degradation. In contrast, when crystalline solvent-cast PHO films were placed in contact with this enzyme, no degradative activity was observed.

Characterization by mass spectrometry of poly(3-hydroxyalkanoates) produced by Rhodospirillum rubrum from 3-hydroxyacids.

The sequence distributions of two microbial copolyesters obtained by fermentation of Rhodospirillum rubrum, grown with 3-hydroxyhexanoic or 3-hydroxyheptanoic acids, were determined by analyzing the oligomers prepared by partial pyrolysis or partial methanolysis of these copolyesters using fast atom bombardment mass spectrometry (FAB-MS). Oligomers up to pentamers were identified in the case of partial pyrolysis and up to tetradecamers in the case of partial methanolysis. The comparison between the experimental and calculated peak intensities of FAB mass spectra allows the calculation of compositions and sequence distributions, which in these copolyesters follow Bernoullian statistics, indicating that they are random terpolyesters.

Biodeuteration of poly(beta-hydroxybutyrate).

The formation of poly(beta-hydroxybutyrate), PHB, by Rhodobacter sphaeroides and Alcaligenes eutrophus was studied using the following carbon sources and solvents: (1), acetate in H2O; (2), D3-acetate in H2O; (3), acetate in 90 to 92% D2O; and (4), D3-acetate in 90 to 92% D2O. The growth of Rb. sphaeroides cultured under condition (2) showed no apparent deuterium isotope effect, while considerably slowed growth in the presence of D2O was observed under conditions (3) and (4). In all cases, the PHB produced under deuterium enriched conditions was of high molecular weight. Interestingly, comparatively high volumetric formation of partially deuterated PHB was obtained using culture condition (4) for A. eutrophus. Fourier transform infrared spectroscopy (FT-i.r.), pyrolysis gas chromatography mass spectrometry (PGC-m.s.), and nuclear magnetic resonance (n.m.r.) were used to establish the extent and distribution of deuterium in the PHB samples produced. Partially deuterated PHB was obtained in each case, using a deuterium enriched culture. Considerable differences in the extent and distribution of deuterium were found between micro-organisms and culture conditions.

Fish and robot dancing together: bluefin killifish females respond differently to the courtship of a robot with varying color morphs.

The experimental integration of bioinspired robots in groups of social animals has become a valuable tool to understand the basis of social behavior and uncover the fundamental determinants of animal communication. In this study, we measured the preference of fertile female bluefin killifish (Lucania goodei) for robotic replicas whose aspect ratio, body size, motion pattern, and color morph were inspired by adult male killifish. The motion of the fish replica was controlled via a robotic platform, which simulated the typical courtship behavior observed in killifish males. The positional preferences of females were measured for three different color morphs (red, yellow, and blue). While variation in preference was high among females, females tend to spend more time in the vicinity of the yellow painted robot replicas. This preference may have emerged because the yellow robot replicas were very bright, particularly in the longer wavelengths (550­700 nm) compared to the red and blue replicas. These findings are in agreement with previous observations in mosquitofish and zebrafish on fish preference for artificially enhanced yellow pigmentation.