ARTIFICIAL SELECTION FOR DEVELOPMENTAL TIME IN DROSOPHILA MELANOGASTER IN RELATION TO THE EVOLUTION OF AGING: DIRECT AND CORRELATED RESPONSES

A wild-type strain of Drosophila melanogaster was successfully selected for both fast and slow larval development. The realized heritabilities (h²) ranged from 0.20 to 0.30 for the fast lines and 0.35 to 0.60 for the slow lines. The selection applied is relevant in relation to the evolution of aging. The longevity of adults, either virgin or mated, was not affected by selection for developmental time, indicating that developmental time is not a causal determinant of life span, thus confirming the results of the studies on environmental effects on aging (Zwaan et al. 1991, 1992). However, adult body weights were higher in the slow developmental lines and lower in the fast lines, relative to the control flies. Furthermore, slow females showed relatively high early fecundity and low late fecundity, as compared with control and fast females. Mated longevities and total lifetime progeny productions were not statistically different. Previous results obtained by other authors from selection experiments on age at reproduction either supported the mutation accumulation or the negative pleiotropy theory of aging (Luckinbill et al. 1984; Rose 1984b). The impact of the reported results on the interpretation of these studies is discussed, and it is noted that direct selection on adult longevity is needed to settle this issue.     

A long-term study on interactions between the Adh and αGpdh allozyme polymorphisms and the chromosomal inversion In(2L)t in a seminatural population of D. melanogaster

The Adh and αGpdh allozyme loci (both located on the second chromosome) showed considerable fluctuations in allele frequencies in a seminatural population of Drosophila melanogaster during 1972–97. Both long-term and short-term fluctuations were observed. The short-term fluctuations occurred within almost all years and comparison of allele frequencies between winters and summers showed significantly higher Adh^S (P < 0.001) and αGpdh^F (P < 0.01) allele frequencies in summers. Frequencies of these alleles were significantly positively correlated with environmental temperature, suggesting the adaptive significance of these allozyme polymorphisms. Frequency changes of the Odh locus (located on the third chromosome) showed no seasonal pattern and were not correlated with environmental temperature. Almost all short-term and long-term increases in Adh^S frequency were accompanied by a corresponding decrease in αGpdh^S frequency (r = –0.82, P < 0.001) and vice versa. Further analysis showed that gametic disequilibria between the Adh and αGpdh loci, which frequently occurred, were due to the presence of inversion In(2L)t located on the same chromosome arm and In(2L)t frequencies were positively correlated with environmental temperature. Gametic disequilibria between Adh and Odh and between Odh and αGpdh were hardly observed. Because In(2L)t is exclusively associated with the Adh^S/αGpdh^F allele combination, the observed correlated response in Adh/αGpdh allele frequencies is (at least partly) explained by hitchhiking effects with In(2L)t. This means that the adaptive value of the allozyme polymorphisms has been overestimated by ignoring In(2L)t polymorphism. Fluctuations in Adh allele frequencies are fully explained by selection on In(2L)t polymorphism, whereas we have shown that αGpdh frequency fluctuations are only partly explained by chromosomal hitchhiking, indicating the presence of selective differences among αGpdh genotypes in relation with temperature and independent of In(2L)t. Frequency fluctuations of αGpdh and In(2L)t are consistent with their latitudinal distributions, assuming that temperature is the main environmental factor varying with latitude that causes directly or indirectly these frequency distributions. However, the results of the tropical greenhouse population show no correlation of Adh (independent of In(2L)t) and Odh allele frequencies with environmental temperature, which may indicate that the latitudinal distribution in allele frequencies for these loci is not the result of selection on the F/S polymorphism in a direct way.     

THE ASSOCIATION BETWEEN FLUCTUATING ASYMMETRY,TRAIT VARIABILITY,TRAIT HERITABILITY,AND STRESS: A MULTIPLY REPLICATED EXPERIMENT ON COMBINED STRESSES IN DROSOPHILA MELANOGASTER

A number of hypotheses have been proposed about the association between developmental stability phenotypic variability, heritability, and environmental stress. Stress is often considered to increase both the asymmetry and phenotypic variability of bilateral traits, although this may depend on trait heritability. Empirical studies of such associations often yield inconsistent results. This may reflect the diversity of traits and conditions used or a low repeatability of any associations. To test for repeatable associations between these variables, multiply replicated experiments were undertaken on Drosophila melanogaster using a combination stress at the egg, larval and adult stages of reduced protein, ethanol in the medium, and a cold shock. Both metric and meristic traits were measured and levels of heritable variation for each trait estimated by maximum likelihood and parent-offspring regression over three generations. Trait means were reduced by stress, whereas among-individual variation increased Fluctuating asymmetry (FA) was increased by stress in some cases, but few comparisons were significant. Only one trait orbital bristle, showed consistent increases in FA. Changes in trait means, trait phenotypic variability, and developmental stability as a result of stress were not correlated. Extreme phenotypes tended to have higher levels of FA but only the results for orbital bristles were significant. All traits had low to intermediate heritabilities except orbital bristle, which showed no heritable variation. Only traits with low heritability and high levels of phenotypic variability may show consistent increases in FA under stress. Overall, the independence of phenotypic variability, plasticity, and the developmental stability of traits extend to changes in these measures under stressful conditions.     

NICOTINE AND α-BUNGAROTOXIN BINDING TO AXONAL AND NON-NEURAL TISSUES1

Abstract— Nicotine binds to homogenates of lobster walking leg nerve (K_d= 1.1 ± 0.3 μm , B_max= 2.4 ± 0.5 nmol/g wet tissue), horseshoe crab leg nerve (K_d= 0.11 ± 0.06 μm , B_max= 1.3 ± 0.6nmol/g), and kidney from 18-month-old rats (K_d= 0.8 ± 0.2 μm , B_max= 23 ± 9 nmol/g). The pharmacological sensitivities of nicotine binding to lobster and horseshoe crab leg nerve homogenates are similar to that of the axonal cholinergic binding macromolecule (ACBM) (Denburg et al., 1972) of lobster leg. nerve membrane, while the binding to rat kidney is sensitive to α-bungarotoxin but not atropine or curare. There was no nicotine binding to rat heart or spleen, or to kidney from younger rats; little or no binding to blue crab nerve or to Torpedo electroplax motor nerve; and little binding (around 0.1 nmol/g) to rat liver. [³H]α-Bungarotoxin bound reversibly (0.17 nmol/g) to lobster leg nerve membrane The implications of these results for the distribution and function of the ACBM, and for the specificity of α-bungarotoxin, are discussed.     

MALE ALTERNATIVE REPRODUCTIVE STRATEGIES IN A MARINE ISOPOD CRUSTACEAN (PARACERCEIS SCULPTA): THE USE OF GENETIC MARKERS TO MEASURE DIFFERENCES IN FERTILIZATION SUCCESS AMONG α-, β-, AND γ-MALES

Three discrete male morphs coexist in Paracerceis sculpta, a marine isopod crustacean inhabiting the northern Gulf of California. Ornamented α-males establish themselves in the spongocoels of intertidal sponges, where females congregate to breed. Smaller β-males, resembling sexually mature females, enter spongocoels by deception, while tiny γ-males invade spongocoels by stealth. Isopods breed year-round, and the operational sex ratio fluctuates widely over short durations. When females are abundant, receptive females accumulate in spongocoels, and these spongocoels are preferentially invaded by β- and γ-males. To test the hypothesis that the density of receptive females affects relative fertilization success among male morphs, individual β- and γ-males, heterozygous for a dominant cuticular pigmentation allele, were placed in artificial spongocoels with an unmarked α-male and densities of one, two, and three unmarked, receptive females. The fertilization success of each male was determined by counting the number of marked and unmarked progeny each female produced. Alpha-males guard females effectively and sire nearly all young when one female is in a spongocoel. The success of β- and γ-males increases, however, and may even exceed the success of α-males when two or three females are present. The regular occurrence of more than one receptive female in the harems of α-males may contribute to the persistence of β- and γ-males in this species.     

PHYLOGENETIC ANALYSIS OF BREEDING SITE USE AND α-AMANITIN TOLERANCE WITHIN THE DROSOPHILA QUINARIA SPECIES GROUP

The Drosophila quinaria group is unusual within the genus in that it comprises both mycophagous and nonmycophagous species. DNA sequence data from three regions of the mitochondrial genome were used to infer relationships among four mycophagous species and three that breed on decaying water plants. Phylogenetic analysis of these species show that breeding in mushrooms and tolerance of high levels of α-amanitin were the ancestral states within the group. Thus, breeding in decaying water plants and intolerance of α-amanitin are derived conditions. We also found that the D. quinaria species group does not comprise separate mycophagous and nonmycophagous clades, but rather that (1) the shift from mushrooms to decaying plants occurred on at least two occasions; or (2) mycophagy reevolved within a lineage that had previously shifted to breeding on plants. The correlation between mycophagy and α-amanitin tolerance is perfect across the species we have examined, indicating that there is no detectable time lag between an ecological shift to a new breeding site and correlated changes in biochemical adaptation. The genetic distance between the mycophagous D. recens and the nonmycophagous D. quinaria indicates that these species split only about 1 M.Y.B.P. In terms of α-amanitin tolerance, D. recens and D. quinaria are typical of other ecologically similar species within the group. Thus, evolutionary changes in α-amanitin tolerance can evidently occur on the order of about 1 million yr. Our data also indicate that, in comparison to other groups of Drosophila, the quinaria species group may be undergoing an adaptive radiation.     

真(鱼岁)(Phoxinus phoxinus(L.))的能量收支各组分与摄食量、体重及温度的关系

在不同的摄食水平(饥饿—最大量)及温度(5—15℃)下,对1—5g的真(鱼岁)的摄食量、排粪量、排泄量、代谢量,生长量及生化组成作了测定。真(鱼岁)的最大摄食量随体重及温度增加而增加。食物能量平均有6.5％损失于粪便中,5.1％损失于排泄物中。摄食代谢随摄食量增加而增加。在同一温度下,特定生长率与摄食量的关系是一减速增长曲线。当摄食不受限制时,生长率随温度增加而增加;当摄食受限制时,生长率随温度增加而下降。鱼体的干物质含量及能量含量随摄食量增加而增加。     

EVIDENCE FOR A SPECIALIZED LOCALIZATION OF THE CHLOROPLAST ATP‐SYNTHASE SUBUNITS α, β, AND γ IN THE EYESPOT APPARATUS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

The eyespot apparatus (EA) of Chlamydomonas reinhardtii P. A. Dang. consists of two layers of carotenoid‐rich lipid globules subtended by thylakoids. The outermost globule layer is additionally associated with the chloroplast envelope membranes and the plasma membrane. In a recent proteomic approach, we identified 202 proteins from isolated EAs of C. reinhardtii via at least two peptides, including, for example, structural components, signalling‐related proteins, and photosynthetic‐related membrane proteins. Here, we have analyzed the proteins of the EA with regard to their topological distribution using thermolysin to find out whether the arrangement of globules and membranes provides protection mechanisms for some of them. From about 230 protein spots separated on two‐dimensional gels, the majority were degraded by thermolysin. Five major protein spots were protected against the action of this protease. These proteins and some that were degradable were identified by mass spectrometry. Surprisingly, the thermolysin‐resistant proteins represented the α and β subunits of the soluble CF₁ complex of the chloroplast ATP synthase. Degradable proteins included typical membrane proteins like LHCs, demonstrating that thermolysin is not in general sterically prevented by the EA structure from reaching membrane‐associated proteins. A control experiment showed that the CF₁ complex of thylakoids is efficiently degraded by thermolysin. Blue native PAGE of thermolysin‐treated EAs followed by SDS‐PAGE revealed that the α and β subunits are present in conjunction with the γ subunit in a thermolysin‐resistant complex. These results provide strong evidence that a significant proportion of these ATP‐synthase subunits have a specialized localization and function within the EA of C. reinhardtii.     

PRELIMINARY ESTIMATES OF THE DOPAMINE β-HYDROXYLASE CONTENT AND ACTIVITY IN PURIFIED NORADRENERGIC VESICLES1

The dopamine β-hydroxylase (DβH) content and activity of large dense-core noradrenergic vesicles purified from bovine splenic nerve were determined using two assay procedures : enzymic activity expressed in Units per mg protein and homospecific activity based on radioimmunoassay expressed in Units per mg DβH antigen. Approximately two-thirds of the total enzyme activity is latent in these vesicles, even after various treatments designed to compromise vesicle integrity. DβH can be completely unmasked by brief treatment with 0.01-0.05% Triton X-100 and activity increases from 0.20 to 0.64 Units per mg vesicle protein. Calculations based on both assay methods suggested that an average of 7% (range 3-15%) of the total vesicle protein was DβH and that the average vesicle contained about 4 molecules of enzyme (range 2-9 molecules). The estimated homospecific activities indicated an average of 25 and 50% (range 18-72%) of the vesicle enzyme was inactive in the various samples using the two antibodies. The vesicle can synthesize up to 30 molecules of noradrenaline/s per molecule of DβH at near optimal substrate concentration, and 60-270 molecules of norepinephrine/s per vesicle. The assumptions used in the various calculations were critically analyzed and, based on the methods employed, it is tentatively considered to be unlikely that there could be more than 5-12 molecules of DβH per vesicle. The possibility that circulating DβH originates primarily, if not exclusively, from the large dense-core vesicle type is considered and the functional implications of the data support the concept of vesicle reuse during several cycles of exocytosis involving a quantal size equal to a fraction of the vesicle transmitter content.     

CHARACTERIZATION OF THE Na+K+-ATPase IN ISOLATED BOVINE ARTICULAR CHONDROCYTES; MOLECULAR EVIDENCE FOR MULTIPLE α AND β ISOFORMS

We have used isoform-specific antibodies against the Na⁺K⁺-ATPase αα1, α2 and α3) and ββ1 and β2) subunit isoforms in order to establish their specific localization in isolated bovine articular chondrocytes. Immunoblotting confirmed the presence of the α1 and α3 isoforms, although α1 expression was significantly greater than α3 as assessed by immunofluorescence confocal laser scanning microscopy and PCR. A similar approach revealed the presence of the β1 and β2 isoforms in chondrocytes, although β2 immunostaining on the plasma membrane was more punctate than β1 which in contrast predominated in a subcellular compartment. The plasma membrane abundance of the Na⁺K⁺-ATPase was found to be sensitive to the extracellular ionic concentration and long-term elevation of extracellular Na⁺concentration significantly upregulated Na⁺K⁺-ATPase density as measured by specific³H-ouabain binding. Our observations suggest that the expression of α3 and β2 is not restricted to excitable tissues as previously reported. The physiological relevance of α3 expression in chondrocytes may be related to its low affinity for intracellular Na⁺in an extracellular environment where Na⁺concentration is unusually high (260–350mm) compared to other cell types (140mm). Glycoproteins and their branched carbohydrates have been implicated in cell recognition events, thus the β2 subunit glycoprotein may allow the chondrocyte to detect changes in its extracellular environment by physically interacting with components of the cellular cytoskeleton and matrix macromolecules.     

EFFECT OF LOW TEMPERATURE ON THE STEREOISOMER COMPOSITION OF β-CAROTENE IN THE HALOTOLERANT ALGA DUNALIELLA BARDAWIL (CHLOROPHYTA)1

Dunaliella bardawil Ben-Amotz & Avron, a β-carotene-accumulating halotolerant alga, was analyzed for the effect of growth temperatures on its pigment content and on the stereoisomeric composition of β-carotene by the use of advanced liquid chromatography and photodiode array detection. Decreasing culture temperature from 30° to 10°C increased the β-carotene content twofold and the ratio of 9-cis to all-trans β-carotene fourfold, with no significant changes in the other cell pigments. The variation of total β-carotene content by temperature was correlated with the integral irradiance received by the algal culture during a cell division cycle, whereas the 9-cis stereoisomer increased over the amount expected by that integration. The massive accumulation of 9-cisβ-carotene within the β-carotene globules is interpreted as indicating that the oily 9-cis stereoisomer protects against the crystallization of all-trans β-carotene at low temperatures.     

GENETICS OF DIELDRIN AND γ-BHC RESISTANCE IN THE HOUSEFLY

A factor for dieldrin resistance in a -BHC selected strain of Uruguayan origin was located in linkage group 4 (Hiroyoshi's numbering system) and showed approximately 40% crossover to the marker ct in F₁ females. Resistance to -BHC is partly due to the same factor. Factors on other linkage groups play an important part in resistance to -BHC, but when studied separately only the fifth linkage group showed a small effect. It is concluded that several factors on different chromosomes must contribute to the total resistance to -BHC.

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Genetik der dieldrin- und -cyclohexan bei der stubenfliege

Zusammenfassung Ein Faktor für Dieldrin-Resistenz in einem -Cyclohexan-selektierten Stamm uruguayischer Herkunft ist in der Koppelungsgruppe 4 (Hiroyoshi's Zählungssystem) gelegen und zeigt bei F₁-Weibchen annähernd 40% Crossing-over gegenüber dem Markierungsgen ct. Die Resistenz gegenüber -Cyclohexan beruht teilweise auf dem gleichen Faktor. Faktoren in anderen Koppelungsgruppen spielen eine wichtige Rolle bei der -Cyclohexan-Resistenz, aber bei getrennter Untersuchung zeigte nur die 5. Koppelungsgruppe eine schwache Wirkung. Es wird daraus geschlossen, daß an der Gesamtresistenz unterschiedliche Faktoren auf verschiedenen Chromosomen beteiligt sein müssen.