Exposure to Lead (Pb2+) Eliminates Avoidance of Pb-Treated Oviposition Substrates in a Dose-Dependent Manner in Female Vinegar Flies.

Female vinegar flies (Drosophila melanogaster) preferentially oviposit eggs on oviposition substrates that decrease larval foraging costs. We tested whether female D. melanogaster would avoid oviposition substrates containing lead (Pb2+), which could potentially decrease offspring fitness. Wild type D. melanogaster were reared on control or Pb-treated medium from egg stage to adulthood and tested for differences in oviposition substrate preference, fecundity (number of eggs laid) and Pb accumulation. Control females laid a significantly lower proportion of eggs on Pb-treated substrates than Pb-treated females. Pb-treated females laid significantly more eggs than control females. Pb-treated adults accumulated significantly more Pb than control-treated adults. These results indicate that Pb exposure disrupts normal oviposition avoidance behaviors, which could increase larval foraging costs for larval offspring. These factors could induce population declines and have cascading implications for the ecosystem.

Intraspecific Genetic Variation for Lead-Induced Changes in Reproductive Strategies.

We aimed to identify genetic variation in the response of reproductive behaviors to lead (Pb2+) exposure. We reared a subset of the Drosophila Genetic Reference Panel (DGRP) inbred lines on control or Pb-treated (500 µM PbAc) medium and tested for differences in copulation latency, copulation duration, and fecundity. Pb exposure decreased fecundity (p < 0.05) and increased copulation duration (p < 0.05) across DGRP lines. We found intraspecific genetic variation in latency, duration, and fecundity in both control and Pb-treated flies, with heritability ranging from 0.45 to 0.80. We found a significant genotype-by-environment interaction for copulation duration (p < 0.05). Genetic correlation matrices revealed significant genetic variation in common between control and Pb-treated flies for each trait (p < 0.05). Our results indicate that intraspecific genetic variation plays a role in Pb susceptibility and emphasize the importance of considering the impacts of variation in susceptibility to Pb pollution.

Accumulation, elimination, sequestration, and genetic variation of lead (Pb2+) loads within and between generations of Drosophila melanogaster.

We examined accumulation, sequestration, elimination, and genetic variation for lead (Pb) loads within and between generations of Drosophila melanogaster. Flies were reared in control or leaded medium at various doses and tested for their Pb loads at different stages of development (larvae, eclosion, newly-eclosed adults, and mature adults). Pb loads were tested using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). We found that D. melanogaster readily accumulated Pb throughout their lifespan and the levels of accumulation increased with Pb exposure in the medium. Wandering third-instar larvae accumulated more Pb than mature adults; this phenomenon may be due to elimination of Pb in the pupal cases during eclosion and/or depuration in adults post-eclosion. The accumulated Pb in mature adults was not transferred to F1 mature adult offspring. Using a set of recombinant inbred strains, we identified a quantitative trait locus for adult Pb loads and found that genetic variation accounted for 34% of the variance in Pb load. We concluded that D. melanogaster is a useful model organism for evaluating changes in Pb loads during development, as well as between generations. Furthermore, we found that genetic factors can influence Pb loads; this provides an essential foundation for evaluating phenotypic variation induced by the toxic effects of Pb.

Asymmetrical positive assortative mating induced by developmental lead (Pb2+) exposure in a model system, Drosophila melanogaster.

Anthropogenic pollutants have the potential to disrupt reproductive strategies. Little is known about how lead (Pb2+) exposure disrupts individual-level responses in reproductive behaviors, which are important for fitness. Drosophila melanogaster was used as a model system to determine the effects of: 1) developmental lead exposure on pre-mating reproductive behaviors (i.e., mate preference), and 2) lead exposure and mating preferences on fitness in the F0 parental generation and F1 un-exposed offspring. Wild-type strains of D. melanogaster were reared from egg stage to adulthood in control or leaded medium (250 µM PbAc) and tested for differences in: mate preference, male song performance, sex pheromone expression, fecundity, mortality, and body weight. F0 leaded females preferentially mated with leaded males (i.e., asymmetrical positive assortative mating) in 2-choice tests. This positive assortative mating was mediated by the females (and not the males) and was dependent upon context and developmental exposure to Pb. Neither the courtship song nor the sex pheromone profile expressed by control and leaded males mediated the positive assortative mating in leaded females. Leaded females did not incur a fitness cost in terms of reduced fecundity, increased mortality, or decreased body weight by mating with leaded males. These results suggest that sublethal exposure to lead during development can alter mate preferences in adults, but not fitness measures once lead exposure has been removed. We suggest that changes in mate preference may induce fitness costs, as well as long-term population and multi-generational implications, if pollution is persistent in the environment.

Multigenerational epigenetic inheritance in humans: DNA methylation changes associated with maternal exposure to lead can be transmitted to the grandchildren.

We report that the DNA methylation profile of a child's neonatal whole blood can be significantly influenced by his or her mother's neonatal blood lead levels (BLL). We recruited 35 mother-infant pairs in Detroit and measured the whole blood lead (Pb) levels and DNA methylation levels at over 450,000 loci from current blood and neonatal blood from both the mother and the child. We found that mothers with high neonatal BLL correlate with altered DNA methylation at 564 loci in their children's neonatal blood. Our results suggest that Pb exposure during pregnancy affects the DNA methylation status of the fetal germ cells, which leads to altered DNA methylation in grandchildren's neonatal dried blood spots. This is the first demonstration that an environmental exposure in pregnant mothers can have an epigenetic effect on the DNA methylation pattern in the grandchildren.

Variations at a quantitative trait locus (QTL) affect development of behavior in lead-exposed Drosophila melanogaster.

We developed Drosophila melanogaster as a model to study correlated behavioral, neuronal and genetic effects of the neurotoxin lead, known to affect cognitive and behavioral development in children. We showed that, as in vertebrates, lead affects both synaptic development and complex behaviors (courtship, fecundity, locomotor activity) in Drosophila. By assessing differential behavioral responses to developmental lead exposure among recombinant inbred Drosophila lines (RI), derived from parental lines Oregon R and Russian 2b, we have now identified a genotype by environment interaction (GEI) for a behavioral trait affected by lead. Drosophila Activity Monitors (TriKinetics, Waltham, MA), which measure activity by counting the number of times a single fly in a small glass tube walks through an infrared beam aimed at the middle of the tube, were used to measure activity of flies, reared from eggs to 4 days of adult age on either control or lead-contaminated medium, from each of 75 RI lines. We observed a significant statistical association between the effect of lead on Average Daytime Activity (ADA) across lines and one marker locus, 30AB, on chromosome 2; we define this as a Quantitative Trait Locus (QTL) associated with behavioral effects of developmental lead exposure. When 30AB was from Russian 2b, lead significantly increased locomotor activity, whereas, when 30AB was from Oregon R, lead decreased it. 30AB contains about 125 genes among which are likely "candidate genes" for the observed lead-dependent behavioral changes. Drosophila are thus a useful, underutilized model for studying behavioral, synaptic and genetic changes following chronic exposure to lead or other neurotoxins during development.

Behavioral effects of chronic exposure to low levels of lead in Drosophila melanogaster.

Through human activity lead has become a serious environmental neurotoxin, known to affect activity levels, attention and both sensory and cognitive function in children. Study of lead would be facilitated by having a model system that could be manipulated easily and quickly. We find Drosophila melanogaster ideal as such, and we have been studying effects of lead on courtship, fecundity and locomotor activity. We raised Canton-S flies from eggs to adult day 6-7 on medium made with lead acetate solution (2-100 microgram/g), or with distilled water, and we measured adult body lead burdens by means of Inductively Coupled Plasma Mass Spectrometry (ICP-MS). To measure courtship effectiveness, five virgin females and five virgin males were transferred into an empty vial and the number of females that mated within 20min was recorded. To measure fecundity, all adult offspring from eggs produced by one female within 12 days of mating were counted. To measure locomotor activity, individual flies were transferred to a grid-labeled petri dish and the number of lines crossed in 30s was counted. The number of females mating within 20min was increased significantly by exposure to 2 or 8 microgram/g lead, and was decreased significantly by exposure to 20 or 50 microgram/g lead. Fecundity was increased significantly by exposure to 2 microgram/g lead, but was unaffected by exposure to 20 microgram/g lead. Locomotor activity was consistently higher for males than for females, and was significantly reduced only by exposure to 50 microgram/g lead, and then only for males. We thus defined for Drosophila a lowest observable effect level (LOEL) of 2 microgram/g lead, which is considerably lower than the doses shown previously to affect this animal. The dose-response curve was biphasic for the number of females mating within 20min, an example of hormesis, a non-linear response that has been reported for low levels of stressors as diverse as pollutants and radiation. We hope from further studies with Drosophila to understand better how lead affects the developing nervous system, and thus ultimately its effects on children.

Effects of chronic lead exposure on the neuromuscular junction in Drosophila larvae.

Long term or chronic exposure to lead is associated with cognitive and other deficits in humans, which may reflect lead-induced changes in synaptic development and function. We believe that Drosophila has great potential as a model system for studying such changes. To test this, we compared the structure of single, identified synapses between identified axons (axons 1 and 2) and muscle fibers (fibers 6 and 7) in untreated 3rd instar larvae, and in larvae reared on medium made with 100 microM lead acetate in distilled water. We used three approaches to examine the motor terminals on muscle fibers 6 and 7 in segment 2: (1) all terminals were stained with an antibody to HRP; (2) only the terminals of axon 1 were stained by injecting biotinylated Lucifer yellow into it; and (3) the regions of the terminal containing synaptic vesicles were stained with an antibody to synaptotagmin, which provides an estimate of "synaptic" terminal area. Lead burdens were determined by inductively coupled plasma mass spectrometry; hemolymph lead levels at the neuromuscular junction were likely to be micromolar. We observed that lead exposure did not significantly affect the average terminal area or the average muscle fiber area, but did significantly affect the uniformity of the matching between muscle area and motor terminal size that normally occurs during development. There was a significant positive correlation between motor terminal size and muscle area in control, but not in lead-exposed larvae. The sensitivity of Drosophila larval synaptic development to lead opens the way to using the powerful genetic and molecular tools available for this system to study the underlying mechanisms of this sensitivity. We would hope that from such an understanding may come strategies for dealing with lead-induced deficits in children.