Fishing directly selects on growth rate via behaviour: implications of growth-selection that is independent of size

Size-selective harvest of fish and crustacean populations has reduced stock numbers, and led to reduced growth rates and earlier maturation. In contrast to the focus on size-selective effects of harvest, here, we test the hypothesis that fishing may select on life-history traits (here, growth rate) via behaviour, even in the absence of size selection. If true, then traditional size-limits used to protect segments of a population cannot fully protect fast growers, because at any given size, fast-growers will be more vulnerable owing to bolder behaviour. We repeatedly measured individual behaviour and growth of 86 crayfish and found that fast-growing individuals were consistently bold and voracious over time, and were subsequently more likely to be harvested in single- and group-trapping trials. In addition, there was some indication that sex had independent effects on behaviour and trappability, whereby females tended to be less active, shyer, slower-growing and less likely to be harvested, but not all these effects were significant. This study represents, to our knowledge, the first across-individual support for this hypothesis, and suggests that behaviour is an important mechanism for fishing selectivity that could potentially lead to evolution of reduced intrinsic growth rates.     

Assembly of microtubules with ATP: evidence that only a fraction of the protein is assembly-competent

Chick brain microtubule protein can be assembled in vitro with ATP, although the extent of assembly is less than that with GTP. The ATP-induced assembly is not the result of generation of GTP by the co-purifying nucleoside diphosphate kinase. Neither an observed increase in the critical concentration nor the phosphorylation of MAP2 can account for the decreased extent of assembly. However, whereas microtubules are formed with both ATP and GTP, incubation with ATP yields additional filaments and polymorphic aggregates. The results demonstrate that of the total protein which can be assembled into microtubules by GTP, about 25-35% is assembled into other structural forms in the presence of ATP.     

Behavioural syndrome in a solitary predator is independent of body size and growth rate

Models explaining behavioural syndromes often focus on state-dependency, linking behavioural variation to individual differences in other phenotypic features. Empirical studies are, however, rare. Here, we tested for a size and growth-dependent stable behavioural syndrome in the juvenile-stages of a solitary apex predator (pike, Esox lucius), shown as repeatable foraging behaviour across risk. Pike swimming activity, latency to prey attack, number of successful and unsuccessful prey attacks was measured during the presence/absence of visual contact with a competitor or predator. Foraging behaviour across risks was considered an appropriate indicator of boldness in this solitary predator where a trade-off between foraging behaviour and threat avoidance has been reported. Support was found for a behavioural syndrome, where the rank order differences in the foraging behaviour between individuals were maintained across time and risk situation. However, individual behaviour was independent of body size and growth in conditions of high food availability, showing no evidence to support the state-dependent personality hypothesis. The importance of a combination of spatial and temporal environmental variation for generating growth differences is highlighted.     

Linear extension rate is independent of colony size in the coral Pocillopora damicornis

Measurements of the skeletal extension rate of branches of the reef coral Pocillopora damicornis showed that the linear extension rate is independent of colony size for colonies from 1.9 to 19 cm in diameter. Analysis of existing data from Western Australia, Samoa, the Great Barrier Reef and Hawaii supports the finding that linear extension is not related to colony size in this species.     

Shared extremes by ectotherms and endotherms: Body elongation in mustelids is associated with small size and reduced limbs

An elongate body with reduced or absent limbs has evolved independently in many ectothermic vertebrate lineages. While much effort has been spent examining the morphological pathways to elongation in these clades, quantitative investigations into the evolution of elongation in endothermic clades are lacking. We quantified body shape in 61 musteloid mammals (red panda, skunks, raccoons, and weasels) using the head‐body elongation ratio. We also examined the morphological changes that may underlie the evolution toward more extreme body plans. We found that a mustelid clade comprised of the subfamilies Helictidinae, Guloninae, Ictonychinae, Mustelinae, and Lutrinae exhibited an evolutionary transition toward more elongate bodies. Furthermore, we discovered that elongation of the body is associated with the evolution of other key traits such as a reduction in body size and a reduction in forelimb length but not hindlimb length. This relationship between body elongation and forelimb length has not previously been quantitatively established for mammals but is consistent with trends exhibited by ectothermic vertebrates and suggests a common pattern of trait covariance associated with body shape evolution. This study provides the framework for documenting body shapes across a wider range of mammalian clades to better understand the morphological changes influencing shape disparity across all vertebrates.     

Transglucosidic reactions of the Aspergillus niger family 3 beta-glucosidase: qualitative and quantitative analyses and evidence that the transglucosidic rate is independent of pH

The hydrolytic and transglucosidic reactions of the Aspergillus niger Family 3 beta-glucosidase were characterized. Michaelis-Menten plots of the rates of aglycone formation were normal (hyperbolic) at low [substrate]. However, at high [substrate] the rates decreased at pH below approximately 5.5 but increased at pH above approximately 5.5. Each decrease or increase took the form of a second hyperbola adjoining the first. Thin layer chromatography, gas-liquid chromatography, and NMR analyses indicated that the substrates became transglucosidic acceptors when present at high concentrations. When pNPGlc and cellobiose reacted as acceptors, the C6 hydroxyl of the non-reducing substrate component reacted to form beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-p-nitrophenol and beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-(1-4)-D-glucopyranose, respectively. The acceptor action accounted for the second adjoining hyperbolas. Rate equations were derived for the production of the aglycone and the transglucosidic intermediate, and these equations described the data very well. Hydrolytic Vmax {Vmax(h)}, hydrolytic Km {Km(h)}, transglucosidic Vmax {Vmax(t)}, and transglucosidic Km {Km(t)} values were obtained by non-linear regression analysis using these equations. Vmax(h) pH profiles were bell shaped with optima between pH 4 and 4.5 but the Vmax(t) values did not change substantially between pH 3 and 7. These differences in the pH profiles explain the decreasing and increasing adjoining hyperbolas since Vmax(t) is lower than Vmax(h) at pH less than approximately 5.5 but higher than Vmax(h) at pH greater than approximately 5.5. The reason for these pH effects is that the value of the hydrolytic rate constant (k3) decreases while the value of the transglucosidic rate constant (k4) does not change between pH 3 and 7. The study also showed that gentiobiose forms by an intermolecular reaction of the C6 hydroxyl of Glc rather than an intramolecular reaction and that an equatorial orientation of the C2 hydroxyl, the presence of a C6 primary hydroxyl and beta-linkages with oligosaccharide acceptors are important for acceptor reactivity.     

End-stabilized microtubules observed in vitro: stability, subunit, interchange, and breakage.

We report a reliable method to prepare, in vitro, microtubules that are stabilized at both ends by axonemal structures, and report studies of their properties. Such "end-stabilized" microtubules neither grow nor shorten over times of several hours when tubulin subunits are present in the surrounding solution. When subunits are removed, the microtubules eventually break. Breakage occurs within a sinuous and flexible region, a few microns in length, that begins at a single point on the microtubule and grows. When breakage does occur, the resulting two free ends shorten very rapidly until the flexible part has depolymerized and the region of straight microtubule is reached. The remainder of the microtubule then shortens at rates comparable to those ordinarily observed in dynamic instability. Formation of the flexible region can be reversed if subunits are added to the buffer prior to breakage. End-stabilized microtubules are a useful tool for studying interactions of molecules with the microtubular wall. They may be a good model for interpreting stabilizing events that happen in the cell. A preliminary study of the effects of microtubule poisons on the wall is presented.     

Ovulation in the perfused ovary in vitro: further evidence that estrogen is not required

The effect of inhibition of estrogen synthesis on ovulation in rat ovaries perfused in vitro with medium without phenol red was examined. The addition of luteinizing hormone (LH, 0.1 microgram/mL) plus 3-isobutyl-1-methylxanthine (IBMX, 0.2 mM) to phenol red-free perfusion medium (M199 + 4% bovine serum albumin) induced ovulation. The number of ovulations was similar to that found in medium containing phenol red. There was a similar increase in estradiol (1, 3, 5 (10)-estratriene-3, 17 beta-diol) levels in the medium in both groups. The addition of 4-hydroxy-4-androstene-3, 17-dione (4-OH-A, 5 microM) to phenol red-free medium blocked the increase in estradiol levels induced by LH + IBMX, but did not prevent ovulation. There was no significant difference in the number of ovulations in the three groups. In conclusion, phenol red in the perfusion medium does not influence ovulation induced by LH + IBMX. Furthermore, an increase in estrogen is not required during the immediate preovulatory period for ovulation to occur.     

Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies

We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.     

Germ cell development in the XXY mouse: evidence that X chromosome reactivation is independent of sexual differentiation

Prior to entry into meiosis, XX germ cells in the fetal ovary undergo X chromosome reactivation. The signal for reactivation is thought to emanate from the genital ridge, but it is unclear whether it is specific to the developing ovary. To determine whether the signals are present in the developing testis as well as the ovary, we examined the expression of X-linked genes in germ cells from XXY male mice. To facilitate this analysis, we generated XXY and XX fetuses carrying X chromosomes that were differentially marked and subject to nonrandom inactivation. This pattern of nonrandom inactivation was maintained in somatic cells but, in XX as well as XXY fetuses, both parental alleles were expressed in germ cell-enriched cell populations. Because testis differentiation is temporally and morphologically normal in the XXY testis and because all germ cells embark upon a male pathway of development, these results provide compelling evidence that X chromosome reactivation in fetal germ cells is independent of the somatic events of sexual differentiation. Proper X chromosome dosage is essential for the normal fertility of male mammals, and abnormalities in germ cell development are apparent in the XXY testis within several days of X reactivation. Studies of exceptional germ cells that survive in the postnatal XXY testis demonstrated that surviving germ cells are exclusively XY and result from rare nondisjunctional events that give rise to clones of XY cells.     

Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy

We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy. In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine the polarity of growth off cellular nucleating centers. We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca++ or colchicine as measured directly by darkfield microscopy. The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell.     

In vivo structural analysis of spliced leader RNAs in Trypanosoma brucei and Leptomonas collosoma: a flexible structure that is independent of cap4 methylations.

The formation of the mRNA 5' end in trypanosomatid protozoa is carried out by trans-splicing, which transfers a spliced leader (SL) sequence and its hypermethylated cap (cap4) from the SL RNA to the pre-mRNA. Previous in vitro studies with synthetic uncapped RNAs have shown that the SL sequence of Leptomonas collosoma can assume two alternate conformations, Form 1 and Form 2, with Form 1 being the dominant one. To gain information about the structure of the SL RNA in vivo, in its protein-rich environment, we have used permeable Trypanosoma brucei and L. collosoma cells for chemical modification experiments. We introduce the use in vivo of the water-soluble reagents CMCT and kethoxal. In contrast to the in vitro results, the Form 2 secondary structure predominates. However, there are chemically accessible regions that suggest conformational flexibility in SL RNPs and a chemically inaccessible region suggestive of protection by protein or involvement in tertiary interactions. Using complementary 2'-O-methyl RNA oligonucleotides, we show that T. brucei SL RNA can be induced to switch conformation in vivo. SL RNA stripped of proteins and probed in vitro does not display the same Form 2 bias, indicating that SL RNA structure is determined, at least in part, by its RNP context. Finally, the methyl groups of the cap4 do not seem to affect the secondary structure of T. brucei SL RNA, as shown by chemical modification of undermethylated SL RNA probed in vivo.     

Association of microtubules and neurofilaments in vitro is not mediated by ATP

Runge et al. [Runge, M.S., Laue, T.M., Yphantis, D.A., Lifsics, M.R., Saito, A., Altin, M., Reinke, K., & Williams, R.C., Jr. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1431-1435] found that mixtures of microtubules and neurofilaments formed a viscous, sedimentable complex when incubated at 37 degrees C for 20 min in the presence of ATP. They did not observe the high viscosities associated with the complex when the incubation was carried out in the absence of ATP. This paper reports an investigation of the roles of time and ATP in the formation of the complex. Microtubules assembled in a mixture containing GTP and neurofilaments prepared from bovine brain remained assembled for a shorter period of time than they did in similar solutions containing no neurofilaments. Adding ATP to the neurofilament-containing solutions, or doubling their GTP concentration, extended the time during which the microtubules remained assembled. These mixtures then became highly viscous. These phenomena resulted from the action of at least two enzymes present in the neurofilament preparation. A GTPase raised the GDP/GTP ratio, in the mixtures in which ATP was absent, to levels sufficient to cause disassembly of the microtubules. When ATP was present, a nucleotide diphosphokinase catalyzed regeneration of GTP from GDP while converting ATP to ADP. This process kept the GDP/GTP ratio low and delayed the disassembly of the microtubules. These results show that the apparent ATP dependence of formation of the microtubule-neurofilament complex observed by Runge et al. is attributable to a GDP-induced disassembly of microtubules rather than to a disruption of microtubule-neurofilament contacts. Those contacts can form in the absence of ATP.     

Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.

We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis.     

Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.     

A molecular marker confirms that the rate of adult maturation is largely independent of the rate of pre-adult development in Drosophila melanogaster

The separation of adult from pre-adult life seen with animals such as Drosophila melanogaster, which are holometabolous and undergo complete metamorphosis, provides the opportunity to examine the contribution of pre-adult rate of development on the rate of maturation and aging of the adult. Recent work has shown that when ambient temperature is used to alter the rate of development there is little effect on adult life span. From this work it has been concluded that the rate of aging is largely independent of the rate of pre-adult development. However, the techniques used to examine life span did not allow for the examination of the earliest events of adult life. Our experimental design used a molecular marker linked to life span as a sensitive measure of determining physiological age. In this way, we were able to evaluate the effect of pre-adult rate of development on the earliest events of adult life. Using ambient temperature to alter both the rate of development in the pre-adult and the rate of aging in the adult independently, we were able to show that it is the ambient temperature at which the adults are living that is the principle determinant of the rate of maturation and aging of the adult. Little effect was seen on the rate of adult maturation in response to an acceleration or a slowing down of the rate of pre-adult development as measured by our molecular marker. These data support the conclusions drawn by others who examined the effect of the rate of development on adult life expectancy. The timing mechanisms at work during pre-adult and adult life appear to be largely regulated separately. If there is such a thing as a physiological clock, it appears to be reset upon eclosion. © 1996 Wiley-Liss, Inc.