Larval competition in Drosophila melanogaster: frequency-dependence of viability

Summary The application of the overfeeding technique (interruption of the competition during larval development) to the study of larval competition in two-strain cultures of Drosophila melanogaster demonstrates the following points: (1) viability is a function of competition time; (2) viability becomes more frequency-dependent as competition time increases; (3) the dynamics of the inner subpopulation (adults that have passed all their development in a crowded condition) and outer subpopulation (adults coming from larvae recovered by interruption of competition) vary with time as regards frequency-dependence; and (4) the wild type strain Oregon is the active agent in competition with the strain cardinal.     

Larval competition in serially transferred populations of Drosophila melanogaster

Summary Experiments showed that larval competition for food is not always the result of decreases in the amount of food available per larva as population density increases. The feeding period during which the larvae try to attain the minimum survival weight may be restricted when food quantity is not limiting. The scramble type competition involves both exploitation and interference components, in degrees which vary with population density.     

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals’ positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.     

Relationship between a metabolic rhythm and emergence rhythm in Drosophila melanogaster

Oxygen consumption and lactic acid dehydrogenase (LDH) activity were determined for Drosophila melanogaster pupae and pharate adults exposed to 12 : 12 or 1 : 23 light-dark (LD) regime. Bimodal circadian fluctuations of oxygen consumption were found in pupae and pharate adults exposed to either LD regime and organisms appeared to demonstrate an anticipatory change in oxygen consumption associated with change in illumination. The oxygen-consumption trend for the entire period spent in the puparium showed a high at the time of emergence, but the diurnal rhythm showed a low at the time of emergence suggesting that emergence occurs at a low in the diurnal cycle. Emergence maximum showed a 3 hr lead over the oxygen-consumption maximum. Changing the LD regime produced similar changes in the phasing of both oxygen consumption and emergence rhythms. LDH activity did not demonstrate a detectable circadian rhythm but did show a steady decrease during pupal and pharate adult development.     

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.     

Variation in abdomen pigmentation in Drosophila melanogaster females

Summary A locus affecting abdomen pigmentation of Drosophila melanogaster females is shown to have a large number of alleles in wild populations. Some of these also affect sternopleural bristle count.     

Nutritional Value-Dependent and Nutritional Value-Independent Effects on Drosophila melanogaster Larval Behavior

Gustatory stimuli allow an organism not only to orient in its environment toward energy-rich food sources to maintain nutrition but also to avoid unpleasant or even poisonous substrates. For both mammals and insects, sugars-perceived as "sweet"-potentially predict nutritional benefit. Interestingly, even Drosophila adult flies are attracted to most high-potency sweeteners preferred by humans. However, the gustatory information of a sugar may be misleading as some sugars, although perceived as "sweet," cannot be metabolized. Accordingly, in adult Drosophila, a postingestive system that additionally evaluates the nutritional benefit of an ingested sugar has been shown to exist. By using a set of seven different sugars, which either offer (fructose, sucrose, glucose, maltodextrin, and sorbitol) or lack (xylose and arabinose) nutritional benefit, we show that Drosophila, at the larval stage, can perceive and evaluate sugars based on both nutrition-dependent and -independent qualities. In detail, we find that larval survival and feeding mainly depend on the nutritional value of a particular sugar. In contrast, larval choice behavior and learning are regulated in a more complex way by nutrition value-dependent and nutrition value-independent information. The simplicity of the larval neuronal circuits and their accessibility to genetic manipulation may ultimately allow one to identify the neuronal and molecular basis of the larval sugar perception systems described here behaviorally.     

Variation between electrophoretically identical alleles at the alcohol dehydrogenase locus in Drosophila melanogaster

A new variant of alcohol dehydrogenase (ADH 7lk) was found in a laboratory stock of Drosophila melanogaster. ADH in this stock had the same electrophoretic mobility as the F variant both on acrylamide and on agar. Activity levels were similar to the levels in F flies at temperature between 15 and 25 C. But while ADH F enzyme is inactivated rapidly at 40 C, ADH 7lk is still active. Also, ADH S is not inactivated at this temperature, but has a far lower activity per fly than ADH 7lk. Genetic analysis showed that the new variant is an allele of the Adh locus.     

Relationship between chromosome content and nuclear diameter in early spermatids of Drosophila melanogaster

We have studied, using light microscopy, the relationship between chromosome content and nuclear diameter in early spermatids of males carrying different combinations of wild-type and compound chromosomes in Drosophila melanogaster. By using these genotypes we have been able to observe spermatid nuclei bearing various numbers of chromosomes ranging from only one sex chromosome and no major autosomes to almost twice the normal chromosome complement. We have found that variations in the chromosome content are accompanied by increasing the variance in early spermatid nuclear diameter; the more gametic classes produced, the higher the variance of nuclear diameters. These results indicate that measuring nuclear diameters in early spermatids represents a useful way to estimate the levels of meiotic non-disjunction and thereby to improve the characterization of lethal or male sterile mutants in which analysis of meiotic chromosome non-disjunction cannot be achieved by conventional genetic methods.     

Genetic and Environmental Effects on the Expression of Peptidases and Larval Viability in Drosophila Melanogaster

The peptidase system in Drosophila melanogaster, consisting of dipeptidase-A, dipeptidase-B, dipeptidase-C and the leucine aminopeptidases, was used as a model to study the adaptive significance of enzyme activity variation. The involvement of the peptidases in osmoregulation has been suggested from the ubiquitous distribution of peptidase activities in nearly all tissues and the high concentration of amino acids and oligopeptides in the hemolymph. Under this hypothesis, larvae counteract increases in environmental osmotic stress by hydrolyzing peptides into amino acids both intra- and extracellularly to increase physiological osmotic concentration. The expression of the peptidases was studied by assaying for peptidase activities in third instar larvae of isogenic lines, which were reared under increasing levels of environmental osmotic stress using either D-mannitol or NaCl. Second and third chromosome substitution isogenic lines were used to assess the relative contribution of regulatory and structural genes in enzyme activity variation. Results indicate that: (1) genetic variation exists for peptidase activities, (2) the effect of osmotic stress is highly variable among peptidases, (3) changes in peptidase activities in response to osmotic stress depend on both genetic background and osmotic effector and (4) peptidase activities are correlated with each other, but these phenotypic correlations depend on genetic background, osmotic effector, and level of osmotic stress. Osmotic concentration in the larval hemolymph is correlated with leucine aminopeptidase activity, but changes in hemolymph osmotic concentration in response to environmental osmotic stress depend on the osmotic effector in the environment. Although these findings suggest that genetic and environmental factors contribute significantly toward the expression of enzymes with similar functions, a relative larval viability study of genotypes that differed significantly in dipeptidase-B (DIP-B) activity revealed that low DIP-B activity did not confer any measurable reduction in larval viability under increasing levels of environmental osmotic stress. These negative results suggest that, either DIP-B does not play a major role in osmoregulation or differential osmoregulation is not related to egg to adult viability in these tests.     

Genetic Variants Affecting Phenoloxidase Activity in Drosophila melanogaster

In Drosophila melanogaster, two new variants affecting the activity of phenoloxidase were found in natural populations at Gomel in Belorussia and at Krasnodar in Russia. Prophenoloxidases, A ₁ and A ₃ , in these variants had the same mobilities on native electrophoresis as the wild type. However, enzymatic activities in their activated states were much lower than in the wild type, whereas the existence of prophenoloxidase proteins was demonstrated. Egg-to-adult and relative viabilities in the variants did not decrease at temperatures between 18 and 29°C. Genetic analyses indicated that the genes showing the phenotype of variants are new alleles of Mox and Dox-3 on the second chromosome.     

The association between malathion resistance and acetylcholinesterase in Drosophila melanogaster

The relationship between the 50% survival time for flies feeding on a malathion-containing medium and the activity of acetylcholinesterase (AChE) was determined for 15 isofemale lines of Drosophila melanogaster. A significant correlation was found (r=0.28, P<0.05), with more resistant lines tending to have a lower level of AChE activity. An association between AChE and malathion resistance was also observed in a selection experiment. The AChE activity decreased in two of two populations selected for malathion resistance. AChE from these populations was altered in kinetic parameters (measured in crude head extracts) and electrophoretic mobility. Although the resistant AChE had a lower activity (V _m) on either a per milligram protein or a per individual basis, its apparent K _m for acetylthiocholine was lower than that of susceptible AChE. Recombination mapping of both low activity and fast electrophoretic mobility localized these traits to the region of the structural locus (Ace) on the third chromosome. The AChE activity of flies heterozygous for a variety of Ace lesions (kindly provided by Dr. W. M. Gelbart) was consistent with this location. The changes in AChE were suggested to have been caused by selection of alleles at the Ace locus.This work was supported by NSERC Grants A5857, G0183, and A0629.     

Genotype-by-Diet Interactions Drive Metabolic Phenotype Variation in Drosophila melanogaster

The rising prevalence of complex disease suggests that alterations to the human environment are increasing the proportion of individuals who exceed a threshold of liability. This might be due either to a global shift in the population mean of underlying contributing traits, or to increased variance of such underlying endophenotypes (such as body weight). To contrast these quantitative genetic mechanisms with respect to weight gain, we have quantified the effect of dietary perturbation on metabolic traits in 146 inbred lines of Drosophila melanogaster and show that genotype-by-diet interactions are pervasive. For several metabolic traits, genotype-by-diet interactions account for far more variance (between 12 and 17%) than diet alone (1–2%), and in some cases have as large an effect as genetics alone (11–23%). Substantial dew point effects were also observed. Larval foraging behavior was found to be a quantitative trait exhibiting significant genetic variation for path length (P = 0.0004). Metabolic and fitness traits exhibited a complex correlation structure, and there was evidence of selection minimizing weight under laboratory conditions. In addition, a high fat diet significantly increases population variance in metabolic phenotypes, suggesting decreased robustness in the face of dietary perturbation. Changes in metabolic trait mean and variance in response to diet indicates that shifts in both population mean and variance in underlying traits could contribute to increases in complex disease.METABOLIC syndrome (MetS) is a complex disease that is promoted by interactions between genetic and environmental effects (O''Rahilly and Farooqi 2006), and seems to be increasing in prevalence in response to a transition from traditional toward Westernized lifestyles (Lee et al. 2004; Schulz et al. 2006). MetS is a constellation of metabolic symptoms including insulin resistance, abdominal obesity, and dyslipidemia, and is predictive of cardiovascular disease and type-2 diabetes (Alberti et al. 2006). The condition has reached epidemic proportions in many Westernized countries (Isomaa et al. 2001; Ford and Giles 2003; Lorenzo et al. 2003; Alberti et al. 2006). Not all individuals are susceptible to the deleterious effects of a Westernized lifestyle, but some individuals are very sensitive to the effects of their environment (Schulz et al. 2006).We argued previously that environmental perturbation contributes to the recent increases in chronic disease in Westernized societies by exposing cryptic genetic variation, a phenomenon that may be particularly evident in metabolic syndrome (Gibson 2009). Increases in complex disease after an environmental shift can be caused by both a change in the population mean or increased variance in a predisposing underlying trait, or endophenotype, causing a larger portion of the population to exceed a disease threshold (Gibson and Reed 2008). Endophenotypes can be molecular, such as rate of uptake of glucose into cells, but also include visible disease covariates, such as body mass. The transition from traditional diets and lifestyles may have perturbed our metabolic homeostasis, thereby promoting increased susceptibility to, and in turn prevalence of, obesity, hyperlipidemia, diabetes, and cardiovascular illness.The complexity of genetic and environmental interactions leads to major challenges in successful disease treatment and prevention strategies, in that it is very difficult to accurately model the relative contributions of nature and nurture to disease susceptibility in a human population. Dietary factors have been demonstrated to interact with specific genetic variants to increase the risk of metabolic disease in humans (Corella and Ordovas 2005; Ordovas 2006; Corella et al. 2009; Warodomwichit et al. 2009), but the relative contribution of overall genotype and environmental effects on human variation is difficult to determine. Modeling population level genotype-by-environment interactions using a model organism like Drosophila can compensate for the research challenges of parameter estimation in human populations.Drosophila share great homology to humans in a number of systems including central metabolism, insulin-signaling pathways, and organs responsible for physiological homoeostasis (e.g., heart, liver, and kidney) (Rizki 1978; Bodmer 1995; Nation 2002; Rulifson et al. 2002; Denholm et al. 2003; Wessells et al. 2004). It has been shown that Drosophila with ablated insulin-producing neurons have elevated hemolymph trehalose levels, considered to parallel a diabetic phenotype (Rulifson et al. 2002). Loss of insulin signaling also restores normal rhythmicity of adult heart rate in old flies (Wessells et al. 2004), providing a link between the obesity and cardiac components of MetS. We have used 146 natural genetic isolates of Drosophila melanogaster to model the relative contributions of genetics, diet, and other environmental effects on the MetS-like phenotypes of larval weight gain, blood sugar concentration, lipid storage, and survival. Individuals from each of these genetic lines were raised on four different diets: their normal lab food, a calorie restricted (0.75% glucose) food, a high (4%) glucose food, and a high fat diet containing (3%) coconut oil.Using this approach, we sought evidence pertaining to two major hypotheses. There are six general types of phenotypic reaction scenarios that a genetically variable population can exhibit in response to an environmental transition: (1) no phenotypic variation in response to genetic or environmental factors, (2) genetic variation in mean phenotype but no change across environments, (3) an additive change in phenotypic mean across genotypes between environments, (4) an interaction effect between genetic and environment leading to a crossing of line means, and (5) a decrease or (6) an increase in variance in the new environment (Gibson and Vanhelden 1997). First, we considered whether the predominant source of metabolic variation within a Drosophila population is genetic, environmental, or the interaction between genetic and environmental effects. Our null was that none of these factors significantly influence weight gain or other phenotypes (scenario 1 above), but it was expected that genetic variation would be prevalent. The more fundamental issue is which of two alternate hypotheses apply: that dietary effects are essentially additive across genotypes (3) or that they are largely genotype dependent (4), possibly also with contributions of behavior and the external environment. Second, we considered whether the transition from a standard laboratory diet to a perturbing diet would change the environmental and/or genetic variance observed in the population: a decrease (5) indicating physiological limitation to the variation or an increase (6) indicating decanalization of the metabolic phenotypes due to loss of physiological buffering.