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.

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.

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.

Speciation in killifish and the role of salt tolerance.

Species pairs whose distributions are tied to environmental conditions provide intriguing candidates for the study of ecological speciation. Here, we examine the role that adaptation to salinity has played in the divergence between two closely related species, Lucania goodei and Lucania parva, whose distributions reflect salinity (L. goodei- fresh water, L. parva- euryhaline). We first tested whether these two species display local adaptation and, subsequently, tested for ecological, genic and behavioural isolation by performing crosses within and between L. goodei and L. parva and raising offspring under various salinities. We found strong evidence for differential adaptation to salinity and also for behavioural isolation where animals preferentially mated with conspecifics over heterospecifics. However, we found no evidence for F1 hybrid inviability. We discuss the general lack of evidence for genic isolation in teleost fish and whether this is a real phenomenon or simply a reflection of experimental design.

Genetic and environmental variation in the visual properties of bluefin killifish, Lucania goodei.

Animals use their sensory systems to detect information about the external environment in order to find mates, locate food and habitat and avoid predators. Yet, there is little understanding of the relative amounts of genetic and/or environmental variation in sensory system properties. In this paper, we demonstrate genetic and environmental variation in opsin expression in a population of bluefin killifish. We measured expression of five opsins (which correlates with relative frequency of corresponding cones) using quantitative, real-time polymerase chain reaction for offspring from a breeding study where offspring were raised under different lighting conditions. Sire (i.e. genetic) effects were present for opsin found in yellow photopigment. Dam effects were present for opsins that create violet, blue and red photopigment. Lighting conditions affected expression of all opsins except SWS2A and mimicked the pattern found among populations. These results highlight the fact that sensory systems are both plastic and yet readily evolvable traits.

Population variation in opsin expression in the bluefin killifish, Lucania goodei: a real-time PCR study.

Quantitative genetics have not been used in vision studies because of the difficulty of objectively measuring large numbers of individuals. Here, we examine the effectiveness of a molecular technique, real-time PCR, as an inference of visual components in the bluefin killifish, Lucania goodei, to determine whether there is population variation in opsin expression. Previous work has shown that spring animals possess a higher frequency of UV and violet cones and a lower frequency of yellow and red cones than swamp animals. Here, we found a good qualitative match between the population differences in opsin expression and those found previously in cone frequency. Spring animals expressed higher amounts of SWS1 and SWS2B opsins (which correspond to UV and violet photopigments) and lower amounts of RH2 and LWS opsins (which correspond to yellow and red photopigments) than swamp animals. The counterintuitive pattern between color pattern, lighting environment, and vision remains. Males with blue anal fins are more abundant in swamps where animals express fewer SWS1 and SWS2B opsins and where transmission of UV/blue wavelengths is low. Understanding this system requires quantitative genetic studies. Real-time PCR is an effective tool for studies requiring inferences of visual physiology in large numbers of individuals.

Intraspecific variation in retinal cone distribution in the bluefin killifish, Lucania goodei.

Studies of visual ecology have typically focused on differences among species while paying less attention to variation among populations and/or individuals. Here, we show that the relative abundance of UV, violet, yellow, and red cones varies between two populations of bluefin killifish, Lucania goodei. Animals from a spring population (high-transmission UV/blue light) have a higher frequency of UV and violet cones and a lower frequency of yellow and red cones than animals from a swamp population (low-transmission UV/blue light). Visual sensitivity does not vary significantly between the populations, but spring animals tend to be more sensitive in the UV/blue wavelengths (360-440 nm) and less sensitive in longer wavelengths (560-600 nm) than swamp animals. The results have two important implications. First, the tight conservation of functional regions of opsin genes across taxa does not imply that visual systems are constrained in their evolution; differential sensitivity can arise through differential expression of cone classes within the retina. Second, intraspecific visual signals in this species may evolve to maximize contrast between the signaler and the background (as opposed to brightness); males with blue anal fins are most abundant in swamp habitats where animals express fewer UV and violet cones.

Enzymatic hydrolysis of oligomeric models of poly-3-hydroxybutyrate.

The mechanism of the enzymatic degradation of poly([R]-3-hydroxybutyrate) (PHB) was investigated by using well-defined model substrates, including both linear and cyclic [R]-3-hydroxybutyrate (3HB) and [R]-3-hydroxyvalerate (3HV) oligomers, with two different PHB depolymerases. The linear and cyclic oligomers containing from 2 to 10 repeating units were hydrolyzed in solutions of the depolymerase isolated from Aspergillus fumigatus and Alcaligenes faecalis, and the rates of hydrolysis and types of products formed were characterized. Both of the depolymerases catalyzed the hydrolysis of the cyclic oligomers (macrolides) which contained more than three 3HB and 3HV repeating units. The degradation reactions of the linear and cyclic 3HB oligomers with the A. fumigatus depolymerase gave similar ratios of monomer-to-dimer products, but PHB itself formed mostly monomer on hydrolysis, indicating that the enzymatic hydrolysis reactions occurred by different mechanisms for these different types of substrates. The results of this study conclusively show that at least the endo mode of polymer hydrolysis occurs with the two enzymes studied, while the A. fumigatus depolymerase was found to utilize both endo and exo modes of hydrolysis to efficiently degrade PHB and 3HB oligomers.

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.

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.

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.

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.

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.

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.

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.

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.