Genotype-by-Environment and Epistatic Interactions in Drosophila Melanogaster: The Effects of Gpdh Allozymes, Genetic Background and Rearing Temperature on Larval Developmental Time and Viability

The possible role of temperature as a component of natural selection generating the latitudinal clines in Gpdh allele frequencies in natural populations of Drosophila melanogaster was examined. Effects of rearing temperature (16 degrees, 22 degrees and 29 degrees) and of Gpdh allozymes (S and F) on larval developmental time and viability were measured. Eight genetic backgrounds from each of three populations (continents) were used to assess the generality of any effects. Analyses of variance indicated significant temperature effects and allozyme-by-genetic background interaction effects for both characters. Viability showed significant genetic background effects, as well as significant temperature-by-allozyme and temperature-by-allozyme-by-population interactions. In general, the S/S genotype was significantly lower in viability than the F/F and F/S genotypes at extreme temperatures (16 degrees and 29 degrees), with no significant differences at 22 degrees. However, each population had a slightly different pattern of viability associated with temperature, and only the Australian population showed a pattern that could contribute to the observed cline formation. Although the same two interactions were not significant for developmental time, examination of the means showed that the S/S genotype had a slightly faster rate of development at 16 degrees than the F/F genotype in all populations (by an average of 0.25 day or 1.1%). The low temperature effect on developmental time is consistent with the clines observed in nature, with the S allele increasing in frequency with higher latitudes. The results for both viability and developmental time are consistent with the interpretation of Gpdh as a minor polygene affecting physiological phenotypes, as indicated by previous work with adult flight metabolism. Finally, it is proposed that the temperature-dependent antagonistic effects of the allozymes on viability vs. developmental time and flight metabolism may be the underlying force giving rise to the worldwide polymorphism.     

Genetic Variability of Flight Metabolism in DROSOPHILA MELANOGASTER. I. Characterization of Power Output during Tethered Flight

The mechanical power imparted to the wings during tethered flight of Drosophila melanogaster is estimated from wing-beat frequency, wing-stroke amplitude and various aspects of wing morphology by applying the steady-state aerodynamics model of insect flight developed by Weis-Fogh (1972, 1973). Wing-beat frequency, the major determinant of power output, is highly correlated with the rate of oxygen consumption. Estimates of power generated during flight should closely reflect rates of ATP production in the flight muscles, since flies do not acquire an oxygen debt or accumulate ATP during flight. In an experiment using 21 chromosome 2 substitution lines, lines were a significant source of variation for all flight parameters measured. Broadsense heritabilities ranged from 0.16 for wing-stroke amplitude to 0.44 for inertial power. The variation among lines is not explained by variation in total body size (i.e., live weight). Line differences in flight parameters are robust with respect to age, ambient temperature and duration of flight. These results indicate that characterization of the power output during tethered flight will provide a sensitive experimental system for detecting the physiological effects of variation in the structure or quantity of the enzymes involved in flight metabolism.     

Genetic Variability of Flight Metabolism in DROSOPHILA MELANOGASTER . II. Relationship between Power Output and Enzyme Activity Levels

The major goal of the studies reported here was to determine the extent to which genetic variation in the activities of the enzymes participating in flight metabolism contributes to variation in the mechanical power output of the flight muscles in Drosophila melanogaster. Isogenic chromosome substitution lines were used to partition the variance of both types of quantitative trait into genetic and environmental components. The mechanical power output was estimated from the wingbeat frequency, wing amplitude and wing morphology of tethered flies by applying the aerodynamic models of Weis-Fogh and Ellington. There were three major results. (1) Chromosomes sampled from natural populations provide a large and repeatable genetic component to the variation in the activities of most of the 15 flight metabolism enzymes investigated and to the variation in the mechanical power output of the flight muscles. (2) The mechanical power output is a sensitive indicator of the rate of flight metabolism (i.e., rate of oxygen consumption during tethered flight). (3) In spite of (1) and (2), no convincing cases of individual enzyme effects on power output were detected, although the number and sign of the significant enzyme-power correlations suggests that such effects are not totally lacking.     

Plastic and Genetic Variation in Wing Loading as a Function of Temperature Within and Among Parallel Clines in Drosophila subobscura

Drosophila subobscura is a European (EU) species that was introducedinto South America (SA) approximately 25 years ago. Previousstudies have found rapid clinal evolution in wing size and inchromosome inversion frequency in the SA colonists, and theseclines parallel those found among the ancestral EU populations.Here we examine thermoplastic changes in wing length in fliesreared at 15, 20, and 25°C from 10 populations on each continent.Wings are plastically largest in flies reared at 15°C (thecoldest temperature) and genetically largest from populationsthat experience cooler temperatures on both continents. We hypothesizethat flies living in cold temperatures benefit from reducedwing loading: ectotherms with cold muscles generate less powerper wing beat, and hence larger wings and/or a smaller masswould facilitate fight. We develop a simple null model, basedon isometric growth, to test our hypothesis. We find that bothEU and SA flies exhibit adaptive plasticity in wing loading:flies reared at 15°C generally have lower wing loadingsthan do flies reared at 20°C or 25°C. Clinal patterns,however, are strikingly different. The ancestral EU populationsshow adaptive clinal variation at rearing a temperature of 15°C:flies from cool climates have lower wing loadings. In the colonizingpopulations from SA, however, we cannot reject the null model:wing loading increases with decreasing clinal temperatures.Our data suggest that selective factors other than flight havefavored the rapid evolution of large overall size at low environmentaltemperatures. However, selection for increased flight abilityin such environments may secondarily favor reduced body mass.     

Human C81 (alpha-gamma) polymorphism: detection in the alpha-gamma subunit on SDS-PAGE, formal genetics and linkage relationship.

The molecular basis of human C81 (alpha-gamma) polymorphism could be elucidated by immunoprecipitation of human C81 allotypes and separation of the alpha-gamma and beta subunits on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. If the C8 molecules were completely reduced, C81 polymorphism was no longer detectable on SDS-PAGE. It is concluded that C81 variation depends on charge rather than molecular weight differences. Four C81 allotypes, the common A and B and two rare allotypes provisionally named A2 and B1, could be distinguished. The rare allotype A1 as detected by isoelectric focusing with subsequent C8 (alpha-gamma)-dependent functional overlay could no longer be visualized on SDS-PAGE. This allotype may therefore be elicited only in the intact C8 molecule. The beta-chain polymorphism named C82, probably also reflecting charge variation of the C8 molecule, could not be detected yet on SDS-PAGE. The distributions of C81 phenotypes and their respective allele frequencies were in good agreement with previously reported data. In the study of 30 families with 100 offspring, no deviation from the rule of at least four codominant alleles at one genetic locus was found. Linkage between C81 gene(s) and PGM1a encoded on chromosome 1 could be confirmed. The following estimates were obtained: (formula; see text) with S theta being the standard error of the maximum likelihood estimate theta. The new technique for allotyping human C81 at the subunit may provide a new tool for the differentiation of qualitative and quantitative variation of the eighth component of human complement.     

Triglyceride pools, flight and activity variation at the Gpdh locus in Drosophila melanogaster

We have created a set of P-element excision-derived Gpdh alleles that generate a range of GPDH activity phenotypes ranging from zero to full activity. By placing these synthetic alleles in isogenic backgrounds, we characterize the effects of minor and major activity variation on two different aspects of Gpdh function: the standing triglyceride pool and glycerol-3-phosphate shuttle-assisted flight. We observe small but statistically significant reductions in triglyceride content for adult Gpdh genotypes possessing 33-80% reductions from normal activity. These small differences scale to a notable proportion of the observed genetic variation in triglyceride content in natural populations. Using a tethered fly assay to assess flight metabolism, we observed that genotypes with 100 and 66% activity exhibited no significant difference in wing-beat frequency (WBF), while activity reductions from 60 to 10% showed statistically significant reductions of approximately 7% in WBF. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection in the triglyceride pool.     

Structural polymorphism of murine complement factor H controlled by a locus located between the Hc and the beta 2M locus on the second chromosome of the mouse

Structural polymorphism of murine factor H protein was demonstrated by using three different methods. 1) By prolonged agarose electrophoresis and immunofixation, factor H protein was visualized in the beta region as a single, distinct protein band in freshly bled EDTA-plasmas from many laboratory and wild mice. Two variants were detected among a large number of tested strains; one, referred to as H.1, moved faster to the anodal region (type strain, BALB/c), and the other, referred to as H.2, moved more slowly to the anodal region (type strain, STR). The F1 hybrid between BALB/c and STR exhibited a combining type of factor H protein, which was observed in each parent. 2) Two-dimensional peptide mapping analysis was carried out with tryptic peptides of these two factor H allotypes. Almost all of the spots in the maps of tryptic peptides were common to both allotypes. However, three distinct spots among the 57 spots detected in the map of tryptic peptides of the H.1 allotypes were not detected in that of H.2 allotype, whereas two spots among the 56 spots in the map of H.2 allotype were unique for this allotype. The F1 hybrid between BALB/c and STR showed a combining type of the map of parent. 3) Alloantisera against each of H allotypes were successfully produced in BALB/c or BALB/c-H.2 (a congenic strain with H.2 allotype) by repeated injection of each purified factor H protein either from the BALB/c or the STR strain. These findings indicated that the observed variants of factor H represent antigenically and structurally distinguishable allotypes. The allotypes of murine factor H protein are controlled by a single codominant locus located between the Hc locus and the beta 2M locus on the second chromosome of the mouse. This was shown by phenotyping the Hc locus and H locus with backcross progenies between A/J (one of strain with H.1) and MoA (one of strain with H.2). The recombination frequency between these two loci was 0.17 +/- 0.046.     

Ambient temperature affects free-flight performance in the fruit fly Drosophila melanogaster

To gain insight into how temperature affects locomotor performance in insects, the limits of flight performance have been estimated in freely flying fruit flies Drosophila melanogaster by determining the maximum load that a fly could carry following take-off. At a low ambient temperature of 15 °C, muscle mechanical power output matches the minimum power requirements for hovering flight. Aerodynamic force production rises with increasing temperature and eventually saturates at a flight force that is roughly equal to 2.1 times the body mass. Within the two-fold range of different body sizes, maximum flight force production during free flight does not decrease with decreasing body size as suggested by standard aerodynamic theories. Estimations of flight muscle mechanical power output yields a peak performance of 110 W kg⁻¹ muscle tissue for short-burst flight that was measured at an ambient temperature of 30 °C. With respect to the uncertainties in estimating muscle mechanical power during free flight, the estimated values are similar to those that were published for flight under tethered flight conditions. Accepted: 5 January 1999     

Rabbit secretory components of different allotypes vary in their carbohydrate content and their sites of N-linked glycosylation

The asparagine-linked glycosylation sites in rabbit high and low Mr secretory components (SC) have been determined for the three known allotypes, t61, t62, and t63. Purified SC polypeptides were subjected to mild periodate oxidation of terminal nonreducing sugars followed by selective reduction with [3H]sodium borohydride, SC polypeptides were further proteolytically cleaved, and the 3H-labeled peptides were isolated and characterized. Both high and low Mr SCs of the three allotypes possess a common glycosylation site at the asparagine residue position 400, whereas the second site, in the amino-terminal domain of SC, was found to be variable: the t61 and t63 allotypes contained a glycosylation site at positions 70 and 90, respectively. Moreover, although the t62 allotype was found to contain a triplet acceptor site (N-X-S) at positions 90-92, analyses showed that less than 30% of the t62 allotype peptides encompassing this region were glycosylated at residue 90. Furthermore, the amino acid sequence of the t61 SC in the region of residues 69-90 varies by 8 and 10 amino acid substitutions when compared with the t62 and t63 allotype sequences, respectively. However, neither the variation in amino acid sequence nor the variation in degree or site of glycosylation measurably affected the non-covalent binding of domain 1 to dimeric IgA.     

Quantitative aspects of proline utilization during flight in tsetse flies

ABSTRACT. The proline concentration of the haemolymph in resting tsetse flies provides a reasonable indication of their total proline content. Estimates of pre-flight proline content obtained on this basis were compared with the proline content of flies that had been flown for different durations to provide an estimate of the rate of proline consumption at different stages of flight. The results indicate that the apparent ability of tsetse flies to continue flight after their proline reserves have been exhausted is an artefact of experimental procedure. It is concluded that the flight capacity of tsetse flies is effectively limited by the magnitude of their proline reserve, although this reserve is capable of being supplemented to some extent by the limited synthetic capacity of the fat body.     

Effect of sex,age and morphological traits on tethered flight of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) at different temperatures

The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is a pest of fruit and vegetable production that has become established in 42 countries in Africa after its first detection in 2003 in Kenya. It is likely that this rapid expansion is partly due to the reported strong capacity for flight by the pest. This study investigated the tethered flight performance of B. dorsalis over a range of constant temperatures in relation to sex and age. Tethered flight of unmated B. dorsalis aged 3, 10 and 21 days was recorded for 1 h using a computerized flight mill at temperatures of 12, 16, 20, 24, 28, 32 and 36 °C. Variations in fly morphology were observed as they aged. Body mass and wing loading increased with age, whereas wing length and wing area reduced as flies aged. Females had slightly larger wings than males but were not significantly heavier. The longest total distance flown by B. dorsalis in 1 h was 1559.58 m. Frequent short, fast flights were recorded at 12 and 36 °C, but long-distance flight was optimal between 20 and 24 °C. Young flies tended to have shorter flight bouts than older flies, which was associated with them flying shorter distances. Heavier flies with greater wing loading flew further than lighter flies. Flight distances recorded on flight mills approximated those recorded in the field, and tethered flight patterns suggest a need to factor temperature into the interpretation of trap captures.     

Contribution of renal secreted complement C3 to the circulating pool in humans

Complement C3 produced within the kidney may be an important mediator of local inflammatory and immunological injury. The overall level of renal C3 production and consequently its contribution to the total circulating C3 level are, however, unknown. This was investigated by using the conversion of C3 from recipient to donor allotype following renal transplantation. The C3 F and S allotypes of 80 consecutive renal donor-recipient pairs (148 individuals) were determined by amplification refractory mutation system analysis. The extent of allotype conversion in C3 F/S mismatched recipients was quantified at different stages after transplantation, using an enzyme-linked immunosorbent assay specific for the HAV 4-1 polymorphism of C3 that is strongly associated with C3F. Twenty-one of the eighty recipients were potentially informative, i.e., were C3 SS recipients of C3 FF or FS donor kidneys. In the early postoperative period, donor-derived C3 (HAV 4-1-positive) was undetectable, increasing to 9.6% of the total circulating C3 at times of acute allograft rejection. When graft dysfunction occurred from causes other than rejection, donor C3 remained undetectable. After stable graft function was attained (3-13 mo after transplantation), donor C3 made up 4.5% of the total circulating C3 pool. Our findings demonstrate that human transplant kidney in the resting state is a significant source of extrahepatic C3. Its heightened local synthesis during rejection episodes suggests a possible pathogenic role for C3 in this immunological process.     

The constraints of body size on aerodynamics and energetics in flying fruit flies: an integrative view

Reynolds number and thus body size may potentially limit aerodynamic force production in flying insects due to relative changes of viscous forces on the beating wings. By comparing four different species of fruit flies similar in shape but with different body mass, we have investigated how small insects cope with changes in fluid mechanical constraints on power requirements for flight and the efficiency with which chemical energy is turned into aerodynamic flight forces. The animals were flown in a flight arena in which stroke kinematics, aerodynamic force production, and carbon dioxide release were measured within the entire working range of the flight motor. The data suggest that during hovering performance mean lift coefficient for flight is higher in smaller animals than in their larger relatives. This result runs counter to predictions based on conventional aerodynamic theory and suggests subtle differences in stroke kinematics between the animals. Estimates in profile power requirements based on high drag coefficient suggest that among all tested species of fruit flies elastic energy storage might not be required to minimize energetic expenditures during flight. Moreover, muscle efficiency significantly increases with increasing body size whereas aerodynamic efficiency tends to decrease with increasing size or Reynolds number. As a consequence of these two opposite trends, total flight efficiency tends to increase only slightly within the 6-fold range of body sizes. Surprisingly, total flight efficiency in fruit flies is broadly independent of different profile power estimates and typically yields mean values between 2–4%.     

Carbohydrate utilization in the thoraces of honey bees (Apis mellifera) during early times of flight

Trehalose levels in the thoraces of honey bees did not change significantly during the first 2–4 min of flight. This effect was seen both for bees flown shortly after removal from the hive and for bees which were flown after a 2 hr starvation period. There was also no detectable activation of the trehalose in isolated mitochondria from bees flown for periods of up to 2 min. However, the glucose content of the thoraces of bees flown shortly after removal from the hive dropped dramatically during the first 15 sec of flight. There was no evidence of a transient increase in the glucose content in the thorax at any of the times studied as would be expected if trehalose were hydrolized. The drop in glucose content at early times of flight was not detected if the bees were starved for 2 hr before flight was started. The changes in fructose content of the thoraces with time were similar to those observed for glucose, but were not statistically significant due to variation among individual bees. The sucrose content of bee thoraces varied greatly and no meaningful conclusions could be reached about how it changed as a function of time of flight.     

Physiology of IgD. V. Enhancement of antibody responses in vivo by allo anti-IgD is due primarily to an indirect effect on B cells

Although responses of BALB/c mice to TNP-Ficoll or TNP-Brucella abortus are usually decreased by injection of allo anti-IgD (anti-Igh-5a) given 1 day before antigen, increased responses are obtained if a lymphokine mixture (SN) containing IL 2 is also injected. Simultaneous injection of anti-IgD and SN 4 days after priming with TNP-KLH induces an increase in antibody production similar to that induced by a second antigen injection. Injected together with a second injection of TNP-KLH at that time, anti-IgD and SN cause a synergistic enhancement of the secondary response. In allotype heterozygous (BALB/c X SJL)F1 mice injected with anti-IgD directed against one allotype, this enhancement of the secondary response is seen predominantly in the alternate allotype, because the IgG response of linked allotype specificity is slightly suppressed by the anti-IgD alone and is less enhanced than the alternate allotype by anti-IgD plus SN. Cells from unprimed heterozygous mice, incubated with anti-Igh-5a in vitro and transferred, together with antigen, to TNP-KLH-primed recipients, cause a much greater enhancement of the IgG responses of the Igb than of the Iga allotype in recipients. If, however, SN is also injected into the recipients, the anti-TNP response of both IgG allotypes is greatly enhanced.     

Rabbit secretory components: identification of a third allotype, t63

A third allotype of rabbit secretory component has been identified. The allotype previously referred to as t62 by our laboratory can now be subdivided into two allotypes, t62 and t63, with alloantisera capable of discriminating between the two. Results of family studies are consistent with a three allele system (t61, t62 and t63) at the t-locus. By SDS PAGE, electrophoretic mobilities of the multiple SC bands for each of the three allotypes are characteristic of the allotype; the apparent molecular sizes of the bands of the t62 allotype are 2 to 3 kDa lower than those for the t61 allotype. The banding patterns of the t61 and t63, although similar, are not identical to each other. Results of serologic cross-reaction studies and of tryptic peptide mapping studies suggest multiple structural differences between the allotypes as well as a closer relationship between t62 and t63 than between either of these allotypes and t61.     

Genetic and Environmental Responses to Temperature of Drosophila Melanogaster from a Latitudinal Cline

Field-collected Drosophila melanogaster from 19 populations in Eastern Australia were measured for body size traits, and the measurements were compared with similar ones on flies from the same populations reared under standard laboratory conditions. Wild caught flies were smaller, and latitudinal trends in size were greater. Reduced size was caused by fewer cells in the wing, and the steeper cline by greater variation in cell area. The reduction in size in field-collected flies may therefore have been caused by reduced nutrition, and the steeper cline may have been caused by an environmental response to latitudinal variation in temperature. No evidence was found for evolution of size traits in response to laboratory culture. The magnitude of phenotypic plasticity in response to temperature of development time, body size, cell size and cell number was examined for six of the populations, to test for latitudinal variation in plasticity. All characters were plastic in response to temperature. Total development time showed no significant latitudinal variation in plasticity, although larval development time showed a marginally significant effect, with most latitudinal variation at intermediate rearing temperatures. Neither thorax length nor wing size and its cellular components showed significant latitudinal variation in plasticity.     

The Beffa form of Simulium soubrense of the S.damnosum complex in Togo and Benin

The Beffa form of Simulium soubrense Vajime & Dunbar, a member of the S. sanctipauli sub-complex of the S. damnosum complex, was found breeding throughout rivers in the Togo-Benin Gap, as far north as 9 degrees 30'N. Its distribution changed with the season. In southern Togo there were seasonal fluctuations in the relative abundancies of the Beffa form and of S. damnosum/S.sirbanum. There was considerable temporal and regional variation in the frequencies of different colour morphs of adult flies. The flies in Benin tended to be darker. Infections with Onchocerca volvulus (Leuckart) appeared to be independent of the host's colour morph category. Larger flies harboured significantly more first stage Onchocerca larvae but no significant relations with fly size were found for second and third stage larvae.     

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.