Lawrence G. Harshman

Laboratory selection experiments using Drosophila: what do they really tell us?

Laboratory selection experiments using Drosophila, and other organisms, are widely used in experimental biology. In particular, such experiments on D. melanogaster life history and stress-related traits have been instrumental in developing the emerging field of experimental evolution. However, similar selection experiments often produce inconsistent correlated responses to selection. Unfortunately, selection experiments are vulnerable to artifacts that are difficult to control. In spite of these problems, selection experiments are a valuable research tool and can contribute to our understanding of evolution in natural populations.

Desiccation and starvation resistance in Drosophila: Patterns of variation at the species, population and intrapopulation levels

A substantial number of Drosophila studies have investigated variation in desiccation and starvation resistance, providing an opportunity to test for consistent patterns of direct and correlated responses across studies and across the species and population levels. In general, responses to laboratory selection for these traits in D. melanogaster are rapid and indicate abundant genetic variation in populations. However, slower responses to selection for desiccation resistance occur in other species including D. simulans. Clines suggest adaptive divergence although specific selection pressures have not been documented empirically. Drosophila species differ markedly in desiccation and starvation resistance and there is also marked variation within species for desiccation resistance that may be linked to local climatic conditions. Laboratory selection experiments on starvation resistance in D. melanogaster suggest that changes in lipid content are largely responsible for resistance variation but this factor may be less important in explaining variation among species. For desiccation, lines with increased resistance show reduced rates of water loss but no changes in the minimum water content that flies can tolerate. Changes in life history traits are sometimes associated with altered levels of stress resistance. Increased starvation resistance is associated with longer development time and reduced early age reproduction in different studies. However, other associations are inconsistent between studies as in the case of stress resistance changing following selection for longevity. Multiple mechanisms may underlie genetic variation in stress resistance and future studies should address the evolutionary importance of the different mechanisms at the population and species levels.

Sperm displacement without sperm transfer in Drosophila melanogaster

In this paper we show that when Drosophila melanogaster females are mated twice, the semen of the second male causes a reduction of the effective number of resident sperm from the previous mating. This is demonstrated by two different kinds of experiments. In one set of experiments, mated females were remated to two different kinds of sterile males, one with normal semen and the other with deficient semen. The effect on the resident sperm was determined from the number of remaining progeny after mating to the sterile male, with the result that the normal semen reduced the amount of resident sperm in comparison with matings to the males with deficient semen. The second set of experiments employed interrupted matings. These experiments were based on the observation that semen is delivered before sperm during the first 5 min of copulation. The second matings were interrupted instantly, 2 min, and 4 min after the initiation of copulation. Compared to the instant interruptions, the two later interruptions had the effect of reducing the amount of resident sperm. The results of these two experiments clearly indicate that a sperm‐incapacitation process plays a role in the well‐documented phenomenon of sperm displacement (last‐male advantage) in this species. Such a process could play a role in sperm displacement in the many cases where the mechanism is unknown.

A cost of reproduction: oxidative stress susceptibility is associated with increased egg production in Drosophila melanogaster

The present study tests the hypothesis that reproduction is correlated with decreased oxidative stress resistance. In numerous species, it has been observed that longevity is negatively correlated with reproduction but the physiological basis of this cost is not well understood. In the present study, female egg production was stimulated by adding live yeast to the surface of Drosophila food. After females were held on yeast-supplemented and unmodified medium for 6-12 days, susceptibility to oxidative stress was measured by exposure to methyl viologen. Added yeast was associated with stress susceptibility of fertile females but not of sterile females. The results of the present study suggest that oxidative stress susceptibility is a physiological cost of reproduction.

A Cost of Reproduction in Drosophila melanogaster: Stress Susceptibility

Abstract.— Little is known about physiological mechanisms that underlie the cost of reproduction. We tested the hypothesis that stress susceptibility is a cost of reproduction. In one test of our hypothesis, Drosophila melanogaster females were exposed to a juvenile hormone analog (methoprene) to stimulate egg production followed by stress assays. A sterile stock of D. melanogaster was employed as a control for reproduction. Exposure of fertile females to methoprene resulted in an increase in female reproduction and increased susceptibility to oxidative stress and starvation (compared to solvent controls). Sterile females did not exhibit a decrease in stress resistance. Mating also stimulated egg production. As a second test of our hypothesis, mated females were compared to virgin females. Mated fertile females were relatively susceptible to oxidative stress, but this relationship was not evident when mated and virgin sterile females were compared. The results of the present study support the hypothesis that stress susceptibility is a cost of reproduction.

INFERENCE OF SPERM COMPETITION FROM BROODS OF FIELD-CAUGHT DROSOPHILA

In field studies of multiple mating and sperm competition there typically is no experimental control over the number of times that a female mates, the interval between matings, or the genetic identity of multiple fathers contributing to a brood. Irrespective of this complexity, high‐resolution molecular markers can be used to assign paternity with considerable confidence. This study employed two highly heterozygous microsatellite loci to assess multiple paternity and sperm displacement in a sample of broods taken from a natural population of Drosophila melanogaster. The large number of alleles present at each of the loci makes it difficult to derive explicit maximum‐likelihood estimates for multiple paternity and sperm displacement from brood samples. Monte Carlo simulations were used to estimate maximum‐likelihood parameters for the distribution of female remating frequency and the proportion of offspring sired by the second or subsequent mating males. Estimates were made based on genotypes scored at two distinct marker loci because they were found to give statistically homogeneous results. Fitting a Poisson distribution of number of matings, the mean number of males mated by a female was 1.82. The sperm displacement parameter estimated from doubly mated females were 0.79 and 0.86 for the two loci (0.83 for the joint estimate). The overall probability that a multiply mated female will be misclassified as singly mated was only 0.006, which indicates that microsatellites can provide excellent resolution for identifying multiple mating. In addition, microsatellites can be used to generate relatively precise estimates of sperm precedence in brood‐structured samples from a natural population.

Life history response of Mediterranean fruit flies to dietary restriction.

The purpose of this study was to investigate medfly longevity and reproduction across a broad spectrum of diet restriction using a protocol similar to those applied in most rodent studies. Age‐specific reproduction and age of death were monitored for 1200 adult males and 1200 females, each individually maintained on one of 12 diets from ad libitum to 30% of ad libitum. Diet was provided in a fixed volume of solution that was fully consumed each day, ensuring control of total nutrient consumption for every fly. Contrary to expectation and precedence, increased longevity was not observed at any level of diet restriction. Among females, reproduction continued across all diet levels despite the cost in terms of increased mortality. Among males, life expectancy exceeded that of females at most diet levels. However, in both sexes, mortality increased more sharply and the pattern of survival changed abruptly once the diet level fell to 50% of ad libitum or below, even though the energetic demands of egg production has no obvious counterpart in males. We believe that a more complete picture of the life table response to dietary restriction will emerge when studies are conducted on a wider range of species and include both sexes, more levels of diet, and the opportunity for mating and reproduction.

Longevity-fertility trade-offs in the tephritid fruit fly, Anastrepha ludens, across dietary-restriction gradients.

Although it is widely known that dietary restriction (DR) not only extends the longevity of a wide range of species but also reduces their reproductive output, the interrelationship of DR, longevity extension and reproduction is not well understood in any organism. Here we address the question: ‘Under what nutritional conditions do the longevity‐enhancing effects resulting from food restriction either counteract, complement or reinforce the mortality costs of reproduction? To answer this question we designed a fine‐grained DR study involving 4800 individuals of the tephritid fruit fly, Anastrepha ludens, in which we measured sex‐specific survival and daily reproduction in females in each of 20 different treatments (sugar : yeast ratios) plus 4 starvation controls. The database generated from this 3‐year study consisted of approximately 100 000 life‐days for each sex and 750 000 eggs distributed over the reproductive lives of 2400 females. The fertility and longevity‐extending responses were used to create contour maps (X‐Y grid) that show the demographic responses (Z‐axis) across dietary gradients that range from complete starvation to both ad libitum sugar‐only and ad libitum standard diet (3 : 1 sugar : yeast). The topographic perspectives reveal demographic equivalencies along nutritional gradients, differences in the graded responses of males and females, egg production costs that are sensitive to the interaction of food amounts and constituents, and orthogonal contours (equivalencies in longevity or reproduction) representing demographic thresholds related to both caloric content and sugar : yeast ratios. In general, the finding that lifespan and reproductive maxima occur at much different nutritional coordinates poses a major challenge for the use of food restriction (or a mimetic) in humans to improve health and extend longevity in humans.

Methionine restriction extends lifespan of Drosophila melanogaster under conditions of low amino acid status

Reduced methionine (Met) intake can extend lifespan of rodents, but whether this regimen represents a general strategy for regulating aging has been controversial. Here we report that Met restriction extends lifespan in both fruit flies and yeast, and that this effect requires low amino acid status. Met restriction in Drosophila mimicks the effect of dietary restriction and is associated with decreased reproduction. However, under conditions of high amino acid status, Met restriction is ineffective and the trade-off between longevity and reproduction is not observed. Overexpression of InRDN or Tsc2 inhibits lifespan extension by Met restriction, suggesting the role of TOR signaling in the Met control of longevity. Overall, this study defines the specific roles of Met and amino acid imbalance in aging and suggests that Met restiction is a general strategy for lifespan extension.

Regulation of juvenile hormone esterase gene expression in the tobacco budworm (Heliothis virescens)

The tissue distribution, developmental control, and induction of juvenile hormone esterase (JHE) mRNA was examined in Heliothis virescens using an 800-base pair fragment of a JHE cDNA clone. Northern hybridization analysis of poly(A)+RNA from fat body and integument of fifth stadium larvae indicated the presence of a single JHE mRNA species having an estimated length of 3 kilobases. On Day 2 of the fifth stadium (L5D2), basal JHE mRNA levels were 3-fold higher in the integument than the fat body, which correlated with the higher specific activity of the enzyme in the integument at this time. However, JHE mRNA levels in the fat body on Day 4 of the fifth stadium were 9-fold higher than on Day 2, while mRNA levels in the integument remained the same. This endogenous increase in JHE mRNA and activity in the fat body occurred at the time of peak hemolymph JHE activity. JHE mRNA was not detected in third stadium larvae which have very low levels of JHE activity. Treatment of L5D2 larvae with the juvenile hormone mimic epofenonane resulted in a 7- and 14-fold increase in the level of JHE mRNA in the integument and fat body, respectively. The mRNA induced in both tissues was of the same estimated length as the constitutively expressed message. The data indicate that the developmental regulation and induction of JHE can occur at the level of mRNA. There is evidence that the fat body secretes more JHE than does the integument and could be the major source of hemolymph JHE.