Download ENVIRONMENTAL STRESS AND ITS EFFECTS ON MUTATION

ENVIRONMENTAL STRESS AND ITS EFFECTS ON MUTATION RATES IN
DROSOPHILA MELANOGASTER
Elizabeth Ann Morgan
A Thesis
Submitted to the Graduate College of Bowling Green
State University in partial fulfillment of
the requirements for the degree of
MASTER OF SCIENCE
December 2005
Committee:
Ron Woodruff, Advisor
Juan L. Bouzat
Dan Pavuk
© 2005
Elizabeth Ann Morgan
All Rights Reserved
iii
ABSTRACT
Ron Woodruff, Advisor.
The role of new mutations in the evolution of adaptation remains controversial. It
is generally believed that the rate of new mutations is too low to confer an immediate
selective advantage. Evolution is thought to occur predominantly through selection
acting upon the standing genetic variation of an organisms genome. In bacteria it has
been shown that stress may cause the activation of mutator genes that increase the
genomic-wide mutation rate. This increase in mutation rate is then selected against when
the selective pressures from the stressor are alleviated. The hypothesis that eukaryotic
organisms may also be able to modulate their mutation rate in response to environmental
stress could explain many aspects of evolution (e.g., the rapid evolution of species during
periods of stress followed by stasis in stable environments). We conducted three
experiments to test the hypothesis that different environmental stressors would increase
either the lethal or the deleterious mutation rate in Drosophila melanogaster. The first
experiment examined the effect of size on the rate of lethal mutations in wild-caught
flies. The remaining two experiments tested the role of vibration as well as interspecific
crowding on both the lethal and deleterious mutation rate. Our results showed a positive
correlation between interspecific crowding and the deleterious mutation rate. Vibration
was correlated with an increase in the lethal mutation rate, and we found a marginally
significant correlation between crowding and the lethal mutation rate. These results are
discussed along with their implications for both evolution and conservation.
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DEDICATION
This thesis is dedicated to my family, both human (Melissa, Daniel and Amber) and canine
(Camille, Chelsea, Gwydion, Julian, Random and Yuri). The humans for endless patience and
the canine for being the only beings in the universe who weren’t completely sick of me before
this was done.
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ACKNOWLEDGEMENTS
I would like to thank the members of my committee, Dan Pavuk and Juan Bouzat, and especially
my advisor Ron Woodruff for all his patience and guidance through this process. I would like to
thank Priti Azad not only for her knowledge of science but for her emotional support when I was
struggling. I would like to thank all the people who read the various forms of this thesis and
gave me feed back: Matt, Jill, Priti and Heather. Tremendous thanks must go to my partner
Melissa Altman. I never would have finished this without you.
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TABLE OF CONTENTS
INTRODUCTION .......................................................................................................................... 1
The Role of Mutation in Adaptation........................................................................................... 2
Stress ........................................................................................................................................... 7
Variation of Mutation Rates Under Stressful Conditions ......................................................... 10
RESEARCH OBJECTIVES AND HYPOTHESIS ...................................................................... 15
Mutation Rates in Wild-Caught Flies ....................................................................................... 15
Vibration ................................................................................................................................... 16
Interspecies Crowding .............................................................................................................. 17
MATERIALS AND METHODS.................................................................................................. 20
The Binscy Assay ..................................................................................................................... 20
Estimations of Deleterious Genomic Mutation Rate Using the Binscy Assay ...................... 21
Estimation of New Recessive Sex-Linked Mutation Rate in Females ................................... 22
The Basc Assay for Recessive Sex-Linked Lethal Mutations .................................................. 22
Culture Conditions .................................................................................................................... 23
Recessive Sex-Linked Lethal Mutation Rates in Wild-Caught Flies ....................................... 23
Vibration ................................................................................................................................... 24
Interspecies Crowding .............................................................................................................. 25
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RESULTS ..................................................................................................................................... 26
Recessive Sex-Linked Lethal Mutation Rates in Wild-Caught Flies ....................................... 26
Vibration ................................................................................................................................... 26
Interspecies Crowding .............................................................................................................. 27
DISCUSSION ............................................................................................................................... 28
Recessive Sex-Linked Lethal Mutation Rates .......................................................................... 28
Trade-offs: Increased Lethal and Deleterious Mutations in Populations......................................
vs. the Possibility of New Beneficial Mutations.................................................................. 32
The High Mutation Rate in Flies Experiencing Interspecies Crowding ................................... 34
CONCLUSION............................................................................................................................. 37
REFERENCES ............................................................................................................................. 38
TABLES ....................................................................................................................................... 52
FIGURES...................................................................................................................................... 57
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LIST OF TABLES
Tables
1a
Page
Comparison of average wing length for offspring from the mating of wild-caught large
and small female D. melanogaster to laboratory stock males ..................................53
1b
Comparison of average body size for F1 generation for the mating of wild-caught large
and small male D. melanogaster to laboratory stock females ..................................53
2
Lethal mutation rate for wild-caught male D. melanogaster..........................................54
3
Lethal mutation rates for D. melanogaster stressed with interspecies crowding or
vibration ....................................................................................................................55
4
Deleterious genomic mutation rates for the X chromosome (Ux), diploid genome (Ud)56
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LIST OF FIGURES
Figure
Page
1
The Binscy mating scheme. ............................................................................................ 58
2
The Basc mating scheme ................................................................................................ 59
3
Changes in mean viability (∆M) over time for deleterious mutations in D. melanogaster
experiencing vibration .............................................................................................. 60
4
Changes in variance (∆V) over time for deleterious mutations in D. melanogaster
experiencing vibration .............................................................................................. 61
5
Changes in mean viability (∆M) over time for deleterious mutations in D. melanogaster
experiencing interspecies crowding.......................................................................... 62
6
Changes in variance (∆V) over time for deleterious mutations in D. melanogaster
experiencing interspecies crowding.......................................................................... 63
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INTRODUCTION
"The phenomena of adaptation is the core of modern evolutionary biology" (Amundsun
1996). Adaptation can either mean the process by which members of a population become better
suited to live and reproduce in the environment in which they are found, or the actual trait that
evolves which helps the organism to survive (Futuyma 1979). This study is concerned with the
former, the processes by which members of a population change to better fit their environment,
specifically the role that new mutations play in the process.
The concept of adaptation is tightly paired with the concept of fitness. Darwin originally
conceived the concept of fitness as an organisms chance of survival in its natural environment
(Darwin 1859). A greater chance of survival meant a greater opportunity to reproduce. Fisher
(1930) later broadened this idea to include all components of an organisms life history, not just
the overall survival rate, including offspring viability, conception rates and mating ability.
Changing environmental pressures may mean that a population must change many different traits
to maintain a steady reproductive rate. However, only an organisms phenotype actually interacts
with its environment. Even though genes are inherited from generation to generation (Dawkins
1976), it is the phenotype that natural selection acts upon in the process of selection (Kimura
1983, Sinervo and Basoio 1996). Adaptation, then, is the process by which a population changes
genotypically and phenotypically to increase fitness in a particular environment.
The phenotype of an organism is the bodily manifestation of the genotype of an organism
(Dawkins 1976). While many factors may affect how a gene is expressed in an individual
organism, such as phenotypic plasticity, epistasis and pleiotropy, ultimately adaptation to new
environmental pressures will involve changes in an organisms genome (Hudson 1996). The
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dominant forces that change the genome are recombination, epistasis (both of which act upon
preexisting mutations), and new mutations (Futuyma 1979).
The Role of Mutation in Adaptation
Spontaneous mutation plays a fundamental role in evolution as the ultimate source of
heritable variation (Kavanaugh and Shaw 2005). While such mechanisms as recombination can
shuffle alleles around producing new phenotypes, selective forces must have heritable variation
on which to act when creating new adaptations. The original source of heritable variations in the
genome is new mutations. New mutations may arise from a variety of sources, including DNA
replication and repair infidelity, spontaneous point mutations, and transposable elements
(Campbell 1987). Any of these may produce changes to the genome that are heritable and may
give rise to new phenotypes on which selection may act.
There are two sources for variation upon which selection can act when selective
pressures change: either the standing genetic variation in the genome or new mutations (Wright
1932, Kimura 1983, Kilpatrick 1996). The standing genetic variation is produced by new
mutations that are then "stockpiled". Most new mutations are recessive (Muller 1962) and once
produced can become incorporated into the genome through drift (Futuyma 1979). This is
assuming that most mutations are neutral or nearly neutral and that the heterozygous state has the
same fitness as the wild-type homozygous state (Kimura 1983, Kreitman 1996). Selection can
then act upon these "stockpiled" alleles (standing genetic variation) when environmental
conditions change. Fishers fundamental theorem of natural selection states: "The rate of increase
in fitness of any organism at anytime is equal to its genetic variation in fitness at that time"
(Fisher 1930). The greater the degree of variability, the greater the chance that a mutation will
already exist that is beneficial. The accumulation of the standing genetic variation within a
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genome is possible because most mutations are considered to be either neutral or only slightly
deleterious in the organisms current environment (Kimura 1984, Drake 1991), and are thus not
selected against. The important concept is the "current environment" of the population. If the
environment changes, exposing the genome to new selection pressures, then selection acting
upon this standing genetic variation is what allows adaptation within a population.
The second way that adaptation may proceed is when new mutations arise, and then
become fixed (Johnson 2000). This fraction of fixations, multiplied by the effects of the fixed
alleles, determines the rate of adaptation (Orr 1998, 2000). Thus, new mutations that arise
during periods of changing environmental conditions (selection pressures) can become
incorporated into the genome and lead to adaptation. New mutations can affect the genome in
several ways; they may be neutral, deleterious, lethal or beneficial. The majority of all new
mutations are either neutral or deleterious (Burgar and Lynch 1977). Neutral mutations do not
affect the phenotype of an organism, and thus neither increase nor decrease the fitness of the
population. Hence, neutral mutations are neither selected for or against. These mutations may
accumulate in a population through drift and contribute to the standing genetic variation. They
may then become evolutionarily important if selective pressures change and they become either
deleterious or beneficial (Lewontin 1974, Kimura 1983)
Since almost all new mutations arise originally in the heterozygous state, new lethal and
deleterious mutational effects are due mostly to the degree of dominance of the mutation
(Kimura 1983). New dominant lethal or deleterious mutations will be rapidly eliminated from
the genome. However, recessive lethal and deleterious mutations may be incorporated or even
selected for, if the heterozygous state confers a selective advantage as in the classic case of
malaria resistance and sickle cell anemia. These new overdominant mutations can be beneficial
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to a population and become stable at a certain frequency within the population due to selection
for the heterozygous phenotype (Allison 2004).
Most deleterious mutations will not, however, impart any new benefits in the
heterozygous state and will be rapidly selected against, even when recessive, if the deleterious
effect is large. However, slightly deleterious mutations may accumulate in the genome,
especially if the effective population is small (Dobzhansky 1950, Kimura 1983). The reduction
in fitness from these slightly deleterious mutations may individually be very slight, but
collectively substantial. The fitness of a population may be greatly impaired by the
accumulation of these slightly deleterious mutations, and this may even lead to extinction
(Charlesworth and Charlesworth 1998). The higher the mutation rate, the faster these mutations
will accumulate impairing fitness (Bell 1997). Over time, populations will evolve a mutation
rate that when balanced by selection leads to a mutation-selection equilibrium (Cooper and
Krawczak 1993). This was first quantified as early as 1930 by Fisher. Simply stated, if U is the
combined rate of deleterious mutation for all loci and s is the average selection against mutants
then the average number of deleterious mutations borne by each individual in a population will
be U/s (Bell 1997). Thus, most populations will exist in any given environment in a state of
equilibrium where the selection pressures will equal the mutation rate (Haldane 1932). A
mutation rate higher than the rate of selection will lead to an accumulation of deleterious
mutations and possible population extinction. Selective pressures greater than the mutation rate
would eventually stop mutations from accumulating, hindering the population in the long term
from adapting to changing environmental pressures.
Beneficial mutations are generally recognized as occurring at a much lower rate than
either deleterious or neutral mutations (Fisher 1930, Lynch et. al. 1999, Erye-Walker et al. 2002,
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Keightley and Lynch 2003), and adaptation usually occurs by the slow accumulation of slightly
beneficial mutations (Futuyma 1979, Bell 1997). Most estimates of mutation rates are based on
deleterious mutations, as the contribution of beneficial mutations is assumed to be negligible
(Fry et al.1999, Lynch et al. 1999). Recently, however, this assumption has come under
scrutiny. Joseph and Hall (2004), for example, reported that 5.75% of all fitness altering
mutations in Saccharomyces cerevisae were beneficial at the end of a mutation-accumulation
(MA) experiment. Also, in MA experiments utilizing Arabidopsis thaliana Shaw et al. (2002)
interpreted their data to conclude that half of all the observed mutations were beneficial. They
then hypothesize that most experiments do not show a greater number of deleterious mutations
when compared to beneficial mutations. What they show is that the effects of the deleterious
mutations are larger and easier to observe. The importance of the ratio of beneficial mutation
rates to deleterious rates is that adaptation is driven mostly by beneficial mutations (Bataillon
2003).
Mutation rates and the proportion of beneficial to deleterious mutations in a genome may
determine if a population can change and adapt to new environmental pressures. A high
mutation rate will bring with it a greater opportunity for beneficial mutations to arise but may
also lead to an accumulation of deleterious mutations that may cause extinctions. This
accumulation of deleterious mutations is what led Sturtevant (1937), among other early
researchers, to assume that in natural populations the selected mutation rate would be the lowest
possible one (for a discussion of the evolution of mutation rates see: Drake 1991, Fry et al.1999,
Houle et al. 1992 and Partridge and Barton 2000).
Estimating mutation rates is a very inexact science. Estimates can be made for both
individual loci and for the entire genome. Since it is impossible to detect neutral mutations
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without sequencing DNA, mutation rates are usually calculated by screening for visible or lethal
mutations or in terms of deleterious mutations affecting one or more fitness traits. The latter is
commonly done through mutation-accumulation experiments, with the mutation rate being
calculated by a procedure initially developed by Bateman (1959) and then later refined by Mukai
(1964). Isogenic lines are established initially from an isogenic base. These lines are then
maintained independently and mutations are allowed to accumulate. Marked cross-over
suppressor chromosomes are used to detect mutations. The mean and variance between
replicates is used to estimate the mutation rate. Using both a single male and female as the
parents each generation minimizes selection. Assuming that the number of mutations per line
after some number of generations is a random variable, different lines will accumulate different
numbers of mutations. Thus, the variance among the lines for a quantitative fitness trait such as
viability will increase over time. Since most mutations are deleterious, the mean value of the
fitness trait will also decline with time. If U represents the mean number of deleterious
mutations that occur per generation, the mean reduction in trait value of a single mutation when
homozygous or hemizygous is represented by s, and if mutational effects are additive across loci,
the rates of decline in overall mean fitness (ΔM) and the increase of variance among the lines
(ΔV) are given by the equations ΔM = Us and ΔV = Us2+ Vs where Vs is the variance among
mutations. This then yields the expression U greater or equal to Δ Ms2/ ΔV. This gives a useful
lower bound on the deleterious mutation rate per genome (Drake et al. 1998). Using this method
a variety of deleterious genomic mutation rates have been estimated for D. melanogaster. These
rates for the diploid genome have varied from as low as 0.04 to as high as 0.9 (Bataillon 2000,
Charlesworth et al. 2004 Chavarrias 2001, Drake et al. 1998, Fry 2003, Fry et al. 1999, Garcia-
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Dorado et al. 1999, Garcia-Dorado et al. 2004, Gong et al. 2005, Keightly and Eyre-Walker
1999, Mukai et al. 1972, Ohnishi 1977).
Most organisms have existed in their current environments for a long time and have
become very well adapted to their environment through natural selection. Even small changes
may affect fitness (Bataillon 2000). Thus, it would be beneficial for mutation rates to remain
low. However, mutations are only beneficial or deleterious within a specific context. Changes
in the selection pressure can alter a previously beneficial mutation to a deleterious one or vice
versa. Changes in selective pressures that affect an organisms fitness are said to create stress
(Lexer and Fay 2005).
Stress
Stress can be defined as any environmental change that reduces the fitness of an organism
(Koehn and Bayne 1989). Stress must always be viewed in context of the organism experiencing
the stress, for what is stressful for one species may be ideal for another or what is stressful during
one stage of an organisms life may become inconsequential in another. Thus, stress may be
thought of as "an environmental factor causing a change in a biological system which is
potentially injurious" (Levitt 1980). The intensity of the stress can then be measured by its effect
on the fitness of the organism under consideration (Hoffman and Parsons 1997).
Environmental stress can come in many forms. The most obvious are abiotic features of
the environment including temperature, climatic factors and chemical components (Lingren and
Laurila 2005). Not so obvious stressors include biotic factors such as competition (both
interspecific and intraspecific), predation, and parasitism (Relyea 2005). Darwin (1859) saw
both intraspecifi and interspecific stress (competition) as the major driving forces behind
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evolution. This is the so often misquoted idea of "survival of the fittest." Haldane (1932) also
extensively discussed the value of competition as an important force in evolution.
Organisms respond to stress on several different levels depending upon the type of stress
encountered. Extreme and rapid changes in environmental conditions may be immediately
lethal, while smaller changes or changes of slower onset may allow the organism time to adapt to
the new situation (Hoffman and Parsons 1997). However, responding and adapting to a stressor
requires an expenditure of energy, which then places an organism at a disadvantage for survival
and/or reproduction. The longer the organism is in the stressed state, the more energy is used.
Even if an organism or population successfully adapts to the new stress, it may then be faced
with other complications due to energy expenditure that may lead to extinction. Thus, the ability
to respond and, if necessary, adapt rapidly to environmental stress, can be an evolutionary
advantage (Hoffman and Parsons 1991).
Many studies have shown that stress response can be positively correlated with genetic
variability in organisms. For example, Crow (1954) observed that variation in DDT resistance
among different strains of D. melanogaster was genetically linked. Hence, Drosophila may have
adapted to this pesticide stress by selecting for either new or standing mutations. In a review
published in 1973, Parsons cited five studies of extreme temperature resistance, four studies of
desiccation resistance, and twelve studies of toxic chemical resistance, all linked to genetic
variability (Parsons 1973). Many of these studies included wild type strains that had evolved to
withstand various environmental stresses in their native habitat. However, several others
included novel stressors (ether, DDT) that would not have been previously encountered and
resistance was considered a by-product of standing genetic variation (Parsons 1973). One
example where response to stress is attributed to new mutations is on warfarin resistance in
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natural populations of rats. Perlz et al. (2005) postulated that up to seven independent mutation
events helped to confer resistance.
Environmental stresses can also act as selective agents that may alter gene frequencies in
populations and consequently cause genetic divergence within and among populations (Hoffman
and Parsons 1991). Many selection experiments in Drosophila have shown that tolerance to
stressors can be dramatically increased over time. Borash and Ho (2001) raised D. melanogaster
populations derived from the same base stock for 90 generations in high larval density conditions
while selecting for larval crowding resistance and for 75 generations in an uncrowded
environment. They found that the larval crowded populations showed a greater resistance to
starvation and greater lipid content than those raised in the uncrowded conditions (Borasch and
Ho 2001). Similarly, Norry and Loeschcke (2002) exposed D. melanogaster to extreme cold
temperatures shock and after 16 generations of artificial selection showed a 25% increase in
adult cold shock resistance in exposed lines compared to a control. To measure desiccation
resistance, Hoffmann and Parsons (1989) exposed flies in a desiccator for ten generations
artificially selecting for desiccation resistance and then relaxing the selective pressure for six
generations. Males from the selected lines showed significantly more resistance to desiccation
than the control lines. While each of these experiments showed that stress resistance could be
selected for, none differentiated between the role played by new mutations versus the standing
genetic pool. For example, studies were not performed on homozygous versus non-homozygous
lines.
Environmental stress may also act to increase the mutation rate or change the expression
of certain genes (phenotypic plasticity) in a genome, giving selection new material on which to
work (Hoffman and Merila 1999, Hoffman and Parsons 1991). Phenotypic plasticity occurs
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when variability in an environmental stimulus leads individuals of the same genotype to develop
into alternative phenotypes (Brakefield 1997). Organisms exposed to a wide range of fluctuating
environmental pressures may evolve the ability to express genes differently. This can be seen in
D. melanogaster when metabolic changes are observed in flies of the same genotype in response
to temperature changes (Bullock 1955). Likewise, Wayne et al. (2005) reared flies collected
from altitude transects on both the north and south canyon wall of "Evolution Canyon" in Israel.
They found that in each of three temperature environments there was significant phenotypic
plasticity for body size in response to temperature (Wayne et al. 2005). Unlike genetic changes,
plastic phenotypic changes will revert back to their original state once the stressor is removed
(Garriel 2005). New mutations would play little or no role in phenotypic changes due to
plasticity.
Variation of Mutation Rates Under Stressful Conditions
Changing the mutation rate in an organism may be of extreme benefit when a population
needs to adapt to new environmental stress and does not have the appropriate standing genetic
variation to do so. As discussed above, normally a balance between mutation and selection
evolves to keep the genome at a steady state in the organisms preferred environment. An
increase in the mutation rate in this system would only be harmful due to the build up of
deleterious mutations. However, beneficial mutations also arise at a greater rate when overall
mutation rates rise, and these beneficial mutations can serve as the basis for adaptation to the
new environmental stress. The ability to increase the mutation rate during stress and then have it
subside to a carrying level once the stress has been adapted to would prevent the continued
accumulation of deleterious mutations, while at the same time increasing the chance of a new
beneficial mutation becoming fixed in the population (Hersh et al. 2004).
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Increased mutation rates during periods of stress have been widely observed in bacteria
(Viser 2002). This is thought to occur largely through the action of mutator alleles (alleles that
increase the mutation rate in the genome). Many mutator alleles are believed to increase the rate
of mutation through interference with the "proofreading" mechanism when DNA is copied. This
interference allows a larger number of mistakes to be incorporated into the genome instead of
being repaired (Drake 1991). Several experiments have shown that if these mutator alleles
become associated with a beneficial allele of another gene then the mutator allele can become
fixed in a population and thus increase the mutation rate of the population (Shaver et al. 2002,
Taddei et al. 1997, Tenaillon et al. 1999). Mao et al. (1997) showed that exposing Lac - E. coli
to a lactose deficient environment lead to an increase of mutator cells from 1/100,000 cells in an
unstressed environment (lactose available) to 1/200 cells in a stressed. These mutators were
linked to a beneficial mutation that that converted Lac- to Lac+ through a frameshift mutation.
Likewise Shaver et al. (2002) showed that in a glucose-limited environment mutator genes were
fixed by hitchhiking with beneficial mutations that arose at other loci. This rise in the mutation
rate allowed for a more rapid adaptation to a novel environment. Once the organism had adapted
to the new environment then the mutator genes would be selected against due to the increase in
deleterious mutations and their associated costs (Travis and Travis 2002). For example, Giraud
et al. (2001) showed that mutation rates in E. coli changed during the colonization of the mouse
gut. Initially strains with a high mutation rate were favored as they allowed for rapid adaptation
to the new environment. As the population became adapted, however, the benefit incurred by the
high mutation rate disappeared and the mutator strains were selected against. This negative
selection was associated with the increase of deleterious mutations in the mutator lines.
Subpopulations that did not decrease their mutation rate once adapted to the new environment
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showed a decrease in survival rate. These experiments with prokaryotes support the hypothesis
that an increased mutation rate can lead to increased fitness in a new, stressful environment.
Increases in mutation rates in eukaryotes due to stress have also been demonstrated. Toxic
chemical stresses and ionizing radiation act as mutagens directly increasing mutation rates
(Hoffman and Parsons 1997). A review by Lingren (1972) showed that temperature extremes in
D. melanogaster (both hot and cold) increased the spontaneous mutation rate. Woodruff et al.
(1983) discuss several mutator systems in wild D. melanogaster populations. Mutators affect
both autosomal and sex-linked chromosomes and clusters of mutations may indicate premiotic
origin of some mutations (Woodruff et al. 1983).
While several mechanisms may account for the increased mutation rate of eukaryotic
organisms under stress, two stand out as the most likely: stress induced error-prone repair
pathways and mobilization of transposable elements (Hoffman and Parsons 1991). DNA repair
systems tend to minimize damage due to external environmental conditions (Friedberg et al.
1995. DNA repair systems, such as the error-prone SOS pathway, may be triggered in stressful
conditions and increase the mutation rate (Hoffman and Parsons 1991). These repair systems
can be acted upon by selection, and studies concerning whether organisms in stressed
environments have a greater efficiency in DNA repair have provided mixed results. Thus, while
El Awadi et al. (2001) and Schmidt-Rose et al. (1999) showed that high temperatures decreased
repair efficiency in human male G0 fibroblasts and human skin cells respectively, Achsa et al.
(2004) showed that repair efficiency was higher for D. melanogaster isofemale lines that were
thermotolerant when compared to thermosensitive lines. It may be that the Drosophila repair
mechanisms have had time to selectively evolve better repair mechanisms than the cells in the
other two studies which do not normally undergo heat stress.
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Transposable elements are mobile segments of DNA that serve as broad-spectrum
mutator elements and are responsible for genetic variation in the host genome (Kidwell 2005).
Unlike recombination events or point mutations, transposable elements are unique in that they
can move genetic material to new sites where it has never existed before and there is evidence
that novel host genes may be mosaic in origin with a mixture of sequences from transposable
elements and host genes (Kidwell 2005). Britten (1996) studied ten examples in eukaryotes
where transposable element insertions affect control of transcription on adjacent genes.
Nekrutenko and Li (2001) have estimated that approximately 4% of the coding sequences in the
human genome are associated with transposable element derived sequences. Across species as
much as 10-90% of the genome is represented by transposable element derived sequences with at
least half of the spontaneous mutations in D. melanogaster arising from insertions of
transposable elements (Finnegan 1992, Kazazian 2004, Nuzhdin and MacCay 1995). The nonLTR retrotransposone L1 that is found in mammals is estimated to account for 1 in every 1200
human mutations (Ostertag and Kazazian 2001) and one half of the human genome may consist
of transposable element sequences (Bowen and Jordan 2002). In Drosophila it has been
hypothesized that transposable elements exist in an equilibrium state balanced between
transposition to new sites and selection eliminating copies. This results in a stable number of
transposable element copies per genome (Charlesworth et al. 1994, Nuzhdin et al.1997). Thus, it
seems logical to assume that an increased mobilization of transposable elements would increase
the mutation rate.
It was observed by McClintock (1984) that heat stress increased the movement of
transposable elements in maize. Paquin and Williamson (1984) made similar observations in
maize, yeast and Drosophila. Lerman et al. (2003) observed that transposable element
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insertions at the hsp 70Ba promoter changed the levels of Hsp 70 produced. Hsp70 is the major
heat-inducible molecular chaperone in D. melanogaster, and elevated levels of Hsp 70 are
usually beneficial for heat tolerance and may be implicated in the resistance to other
environmental stress factors (Sorensen et al. 2003). While most transposable element movement
would be deleterious due to the disruption of normal gene function, Aminetzach et al. (2005)
have observed that the insertion of a transposable element of the Doc family (Doc 1420) actually
appears to confer resistance to the organophosphate pesticide azinphos-methylphosphate. A
similar observation has shown that a Doc insertion in 5' flanking region of the gene Cyp6g1
greatly increased the resistance to DDT in both D. melanogaster and D. simulans. The
transposable element is correlated with increased transcript abundance of Cyp681 and is
hypothesized to have moved transcriptional information between related genes on different
chromosomes (Schlenke and Begun 2004). These observations support a model developed by
Edwards and Brookfield (2003) which showed that in a fluctuating environment some
transposable element insertions could be beneficial. Likewise, transposable elements may exert
major influence on the genome of the host through creating a plasticity to evolve novel genes in
response to new environmental situations (Cayp et al. 2000, Kidwell and Lisch 1997, 2000).
In summary, the ability to increase and decrease the mutation rate in response to
environmental stress in eukaryotic organisms could be beneficial. This ability would allow a
population to generate more diversity upon which selection could act during times of stress when
adaptation could become crucial. Yet, it would also allow the mutation rates to drop down to
carrying levels during times of environmental stability so as to reduce the accumulation of
deleterious mutations.
15
RESEARCH OBJECTIVES AND HYPOTHESIS
Three experiments with the model organism Drosophila melanogaster have been
performed to determine whether stress increases mutation rates in eukaryotic organisms. The
first study, which utilized the Basc assay, tested the hypothesis that small wild-caught flies
would have a significantly higher lethal mutation rate than large wild-caught flies. In the other
two studies the Binscy assay was used to determine if two different stressors (interspecies
crowding and vibration exposure) increased the rate of either lethal or deleterious mutations in
laboratory stocks. We hypothesized that the presence of stressors, such as interspecies crowding
or vibration, would significantly increase mutation rates.
Mutation Rates in Wild-Caught Flies
Reduced body size can often be an indication that an organism has been stressed (Walters
et al. 1996). For example, ponies exposed to low caloric and nutritional content diets on the
island of Assateague are significantly smaller than genetically similar ponies bred from the same
base stock in more optimum conditions (Draper 2000). Lefranc et al. (2000) showed that
starvation had a direct effect on adult body size in D. melanogaster, with starved flies being
significantly smaller. Robertson (1960) also showed a similar correlation in D. melanogaster
with body size dependent upon the nutritional content of various growth media. If the hypothesis
that stress increases mutation rate is correct, then smaller flies produced by environmental stress,
such as low nutrition in nature, should have a higher mutation rate than larger flies. To test this
hypothesis, Ivannikov and Zakharov (1996) and Ivanov and Ivannikov (1997) divided wildcaught male flies visually for size and then determined the mean weight of each group (small:
0.26mg - 0.53mg, intermediate: 0.78mg - 0.79mg and large: 0.74 - 0.9mg). The rate of new
16
lethal mutations on the X chromosome in the wild-caught males in each size group was then
calculated by crossing the wild-caught males with Basc females (see Materials and Methods).
Smaller flies had significantly higher lethal mutation rates than larger flies (Ivanov and
Ivannikov 1997, Ivannikov and Zakharov 1996). It is important to confirm that these results are
also true for flies from additional natural populations. In this experiment on the effect of size on
mutation rates, male flies were visually divided between small and large, measured, and then
each group assayed for differences in mutation rate. We were seeking to prove or disprove the
original hypothesis of Ivannikov and Zakharov (1996) that small sized flies have a greater
mutation rate. Both small wild-caught males and females were also crossed with laboratory
stocks and the size of the F1 generation flies were measured to determine if the small size was
due to environmental stress or genetic factors influencing size.
Vibration
To test the influence of vibrational stress on mutation rates, D. melanogaster had been
treated with ultrasonic vibration (greater than 20,000 Hz) throughout their lifecycle. Recessive
and dominant lethal mutation rates are increased by these treatments (Kato 1966b). Likewise
Saitta and Crenshaw (1971) showed that much lower frequency vibration (100 Hz) increased the
number of inviable eggs produced from females crossed with treated males as compared to
control males. More recently, while simulating the environmental stresses caused by NASA
space shuttle flight, Thompson and Woodruff (unpublished) showed that D. melanogaster
exposed to vibration frequencies from 20Hz - 2KHz (in a pattern simulating vibration during a
NASA shuttle lift-off) from 150Hz - 1KHz showed a significant increase in non-disjunction
events. However, flies exposed to similar vibration did not show an increase in chromosomal
breakage. Experiments on the effect of both noise and vibration on sister chromatid exchange in
17
humans concluded that aerospace workers exposed to whole-body vibration and noise from a
variety of sources exhibited a significant increase in sister chromatid exchange (Silva 1999a).
Similarly, airforce pilots exposed to the high-intensity, low-frequency noise, whole-body
vibration, and g-force experienced during aircraft maneuvers also showed a higher frequency of
sister chromatid exchange (Silva 1999b). In an attempt to separate the causes of the increased
sister chromatid exchange, Silva et al. (2002) exposed mice to both low frequency noise (90 109dB) and to a combination of low-frequency noise and whole-body vibration (12 Hz). Their
results showed that low-frequency noise alone did not increase sister chromatid exchange while
the combination of low-frequency noise and whole-body vibration did. In this experiment, we
exposed D. melanogaster to whole-body vibration at 400rpm (6.67Hz) every ten minutes for the
entire lifecycle (except for a brief mating period) to determine if the exposure to low-level
vibration would increase the mutation rate.
Interspecies Crowding
The environmental stress of crowding occurs with considerable frequency in the natural
environment, especially in social animals such as birds (Fairbrother et al. 2004) and insects
(Mueller et al. 2004) and animals forced into association through habitat fragmentation (Holmes
1996) Both interspecific and intraspecific crowding would be experienced as organisms compete
both among themselves and with other organisms striving to utilize the same resources.
Crowding often plays a large role in population behavioral dynamics (migration, mate choice),
but can it influence populations on the genetic level? For example, larval crowding has been
observed to drive phenotypic variation in D. melanogaster. Imasheva and Bubliy (2003)
observed the effects of low, intermediate and high larval crowding on four morphological traits:
thorax and wing length, sternopleural and abdominal bristle number. Under high larval densities
18
all four morphological traits increased in phenotypic variance. The authors stated that the
increase in phenotypic variance could be due to an increase in additive genetic variance, but did
not pursue the role of new mutation.
If the increase in phenotypic variation under stress is due to an increase in additive
genetic variation, then observable variation should respond to selection. Several selection
experiments have shown a positive response for resistance to the detrimental effects of crowding.
Crowding is clearly detrimental. As an example, Joshi et al. (1998) showed that adult crowding
in D. melanogaster increased the incidence of mortality, while decreasing fecundity
significantly. Populations selected for adaptation to high adult densities also had significantly
lower mortality rates. Similarly, Borash and Ho (2001) showed that populations of D.
melanogaster raised under conditions of extreme crowding when selected for viability showed a
greater resistance to starvation and higher lipid storage than control populations. While both of
these studies showed that adaptability to crowded conditions could be selected for, neither of
them addressed the question of whether selection was working on standing genetic variation or
new mutations.
In this study, populations of D. melanogaster were placed in crowded conditions with a
very similar species in the melanogaster species subgroup: D. simulans. Both of these species
are very similar in their size and nutritional needs and can only be differentiated phenotypically
by the male genitalia (Ashburner 1989). Phylogenetic reconstruction of the evolution of the
melanogaster species subgroup estimates that D. melanogaster and D. simulans may have shared
a common ancestor as recently as 2.5 million years ago (Ashburner 1989). In the wild, D.
simulans has a very similar geographical distribution to D. melanogaster and appears to avoid
direct competition by micropartitioning of the environment (Kawanishi and Watanabe 1981).
19
However, in the laboratory environment they produce hybrids, though only one sex survives and
is sterile (Ashburner 1989). Thus, D. simulans is a good candidate to directly stress D.
melanogaster in the laboratory environment through crowding in a shared environment with
limited resources. The purpose of this experiment was to examine whether interspecies
crowding would increase the rate of new mutation as a response to environmental stress .
20
MATERIALS AND METHODS
The Binscy Assay
In the synthesis of the Binscy assay stocks we used a mating scheme modified from Muller
and Oster (1963) (see Gong, et al., 2005 for a discussion of this mating scheme). Binscy is a
balancer X chromosome with the B (Bar eyes, dominant) and y (yellow body, recessive)
mutations, plus multiple inversions that eliminate recombination of the X chromosome in
females. Gong et al. (2005) confirmed the balancing ability of the Binscy X chromosome by
observing no recombination among 622 progeny from w m f/Binscy females as opposed to 206
recombinants recovered among 584 progeny from w m f / + + + females. C(1;YS)oc ptg is a
combination of the X and the short arm of the Y chromosome, with the oc (ocelliless female,
homozygous female sterile) and the ptg (pentagon, thoracic trident dark) recessive mutations.
RYL is the long arm of the Y chromosome in the shape of a ring. The Binscy/RYL males are
sterile because of the missing male fertility factors on the short arm of the Y chromosome, and
C(1;YS)oc ptg/C(1;YS)oc ptg females are sterile because of the homozygous oc mutation. For
each generation, one Binscy/C(1;YS)oc ptg female was mated with one C(1;YS)oc ptg/RYL male
(Figure 1). New deleterious mutations will accumulate over the generations on the Binscy
balancer X chromosome in the Binscy/C(1;YS)oc ptg females. Since C(1;YS)oc ptg homozygous
females and Binscy/RYL males from this mating scheme are sterile, new deleterious mutations
that occur on the X chromosome of the C(1;YS)oc ptg/RYL males are in a hemizygous state and
are eliminated by selection. To the extent that deleterious mutations might temporarily be
present on the C(1;YS) oc ptg, the calculation of Ud (diploid mutation rate) will be decreased.
However, deleterious mutations that occur on the Binscy X chromosome in Binscy/C(1;YS)oc
21
ptg females are buffered from selection because mutations are maintained as heterozygotes
against wild-type alleles on the C(1;YS)oc ptg chromosome. So, over time, deleterious mutations
will accumulate on the Binscy chromosome but are not expected to remain on the C(1;YS)oc ptg
chromosome. By comparing the viability of Binscy/RYL males (where mutations are
accumulating) to the viability of C(1;YS)oc ptg/RYL males (where mutations are not
accumulating), the rate of new mutations on the X chromosome can be determined using the
method of Bateman (1959) and Mukai (1964).
Estimations of Deleterious Genomic Mutation Rate Using the Binscy Assay
The deleterious genomic mutation rate was calculated using a method published by Gong et
al. (2005) that followed the Bateman - Mukai technique (Drake et al. 1998, Lynch and Walsh
1998). The accumulation of deleterious mutations over time will lead to a predicted, steady
reduction in mean viability and an increase in variance between lines. The rate of decline in
mean variability (∆M) and the rate of increase in variance of viability among lines (∆V) can be
estimated by regression analysis. If one assumes that spontaneous mutations are distributed on
the X chromosome according to a Poisson distribution, ∆M and ∆V can be expressed as:
∆M = MsUx
∆V = (Ms2 + Vs)Ux
Rearranging these two equations gives
Ux = ∆M2/∆V,
where Ms and Vs are the mean and variance of s (the effect of a mutation on viability) and Ux is
the mean number of deleterious mutations on the X chromosome in one generation. Since the
Binscy assay estimates the haploid mutation rate using the X chromosome and the X
22
chromosome contains about 15.97% of the genes in the haploid genome in D. melanogaster, the
estimated diploid mutation rate (Ud) is two times Ux, divided by 15.97% (Gong et al. 2005).
One major problem with determining mutation rates is the lack of good controls. In D.
melanogaster a variety of comparative controls have been used including the original base stock,
the most fit lines at the end of the experiment, and base stocks frozen at the beginning of the
experiment (Gong et al. 2005). The Binscy assay has the advantage of using sibling controls
derived from the same original base stock. The mating of one sibling pair each generation keeps
the population small, eliminating selection.
Estimation of New Recessive Sex-Linked Mutation Rate in Females
A new lethal mutation in the Binscy X chromosome was counted when a line had no
Binscy/RYL males for three generations or more. The summation of the total number of lines
screened in each generation is the total number Binscy X chromosomes assayed. Division of the
number of lines showing no Binscy/RYL males by the total number of lines assayed gives the
new lethal mutation rate.
The Basc Assay for Recessive Sex-Linked Lethal Mutations
The Basc X chromosome is a balancer that contains the multiple inversions In(1)scSIL sc8R
+ S, sc8 scS1, the visible dominant marker Bar (B) eyes, and the recessive mutation white-apricot
(wa) eyes. The Basc/ Y males and homozygous females are viable and fertile and the Basc
chromosome, due to the inversions, suppresses crossing over in the X chromosome. A single
wild-caught +/Y male is crossed with one or more Basc/Basc females and F1 progeny (/Basc
female and Basc/Y male) are then crossed to produce F2 offspring with four different phenotypes
(Figure 2). A new, lethal mutation in the X-chromosome of the gametes of the original wild-
23
caught male will be transmitted through the F1 female to the F2 generation. The presence of a
new, lethal mutation can then be detected by the absence of wild type males in the F2 generation.
A lethal mutation is declared if twenty or more Basc males are observed but no wild-type males
in the F2, and, if needed, F3 progeny. Scoring the number of observed lethal mutations against
the overall number of gametes (vials) examined gives the rate of new lethal mutations (Lee et al.
1983, Woodruff et al. 1985).
Culture Conditions
All flies were reared in standard plastic vials with cornmeal-molasses-agar medium
seeded with a few grains of live yeast. The flies were incubated at 25oC and the adults were
handled under ethyl ether anesthesia.
Recessive Sex-Linked Lethal Mutation Rates in Wild-Caught Flies
Male flies were collected over the course of four summers from 2002 - 2005 in various
locations in the Greater Toledo, Ohio area. Large flies and small flies are relative terms because
flies must be considered in the context of the population from which they are collected. As a
general rule flies larger than 0.25mm where considered large and flies smaller than 0.25 were
considered small. The wild-caught males were then crossed individually with Basc females.
The F1 siblings were then paired and allowed to mate, and the F2 generation was scored for the
absence of wild-type males (indicating a lethal mutation in the original wild-caught males germ
line). A total of 5419 gametes were observed (2556 from big males and 2863 from small). The
significance of the difference between lethal mutations in large and small wild-caught males
were then calculated using a 2 x 2 contingency test. To test whether small flies are small
because of stress or because of their genetic make-up, the wing size of wild-caught female flies
24
were measured and small and large wild-caught females were bred to laboratory stocks. The
wing size of the F1 females were then measured. We also bred the large and small males used in
this experiment to laboratory stock females of approximate equal size and recorded the body size
of the F1 generation. If stress, such as poor nutrition, is the cause of reduced body size then the
F1 generation should show no significant differences in progeny size from small or large parents.
However, if the reduction in body size is due to the genetics of the parents, then the F1 progeny
from the small flies will be significantly smaller than from the large flies.
Vibration
To begin the experiment, 200 lines were set up using one pair of (1; YS) oc ptg / RYL
male and one Binscy/ C(1; YS) oc ptg female in each vial. One hundred of these vials were
placed on a Junior Orbit Shaker from Lab-line Instrument Inc. and shaken at 400 rpm (6.67 Hz)
for 10 seconds every 20 minutes for their entire life cycle. The other 100 lines were kept in the
same conditions but not vibrated as a control. Since there was only one shaker available, backup
vials were not set up and a line was considered extinct when the original line expired. To ensure
that eggs had been laid and that inviability of a line was not caused by the vibration hindering the
mating process or egg laying, the vibrated flies were allowed to lay eggs for five days, then the
vials were cleared and placed on the shaker. Offspring were counted for seven days from initial
enclosure. Only lines that were still viable at the end of generation 12 were used to calculate the
genomic mutation rate. Both the genomic mutation rate and the lethal mutation rate was
calculated in the same manner as it was for the crowded flies.
25
Interspecies Crowding
To begin the experiment, 200 lines were set up using one C(1; YS) oc ptg / RYL male and
one Binscy/ C(1; YS) oc ptg female in each vial. Three male/female pairs of D. simulans with
the wa (white-apricot eyes) mutation were then added to each of 100 lines with the other 100,
without D. simulans, kept as a control. This ratio of D. melanogaster to D. simulans was
necessary to insure that D. simulans had viable larva in each vial. Duplicate vials for each
original vial were set up using siblings and a line was only considered extinct when both lines
had expired. For each generation a new pair of D. melanogaster was selected and three pairs of
D. simulans of equal age from a stock were added at the same time to ensure equal egg laying
time and equivalent larval age. The vials were cleared after five days and offspring were
counted for seven days from initial adult enclosures. Only lines that were still viable at the end
of generation eleven were used to calculate the deleterious genomic mutation rates. The ratio of
Binscy males produced to total D. melanogaster males in each generation was used to calculate
the deleterious genomic mutation rate. Lines that showed no Binscy/RYL males after three
consecutive generations were also considered to have a new lethal mutation on the Binscy X
chromosome. The ratio of non-Binscy/RYL lines to lines still producing Binscy/RYL males was
used to calculate the spontaneous lethal mutation rate.
26
RESULTS
Recessive Sex-Linked Lethal Mutation Rates in Wild-Caught Flies
The first thing that needed to be determined for the wild-caught flies was if the observed
difference in male size was due to stressed conditions or to genetics. Mating both large and
small wild-caught females/males with laboratory stock males/females showed that the observed
difference in the wild-caught males was probably environmental and not genetic. In both
crosses, using a one way ANOVA test, the F1 offspring size was not significantly different with
P = 0.529 for wild-caught males and P = 0.355 for wild-caught females (Table 1a,b).
The results of the large/small wild-caught male fly comparison (Table 2) show a total for
all four years of 16/3066 (0.52%) new lethal mutations per chromosomes assayed in the small
flies as opposed to 8/2554 (0.31%) in the large flies (P = 0.234).
Vibration
The vibrated flies showed a significant difference in the number of new lethal mutations
per chromosome assayed. The vibrated male flies had eight new mutations for 1116
chromosomes assayed (0.72%) while the control had two for 1250 chromosomes (0.16%)
assayed. This gives a P value of 0.0380 (Table 3).
For the MA accumulation experiment used to calculate the genomic wide deleterious
mutation rate, the control showed a ∆M of 0.00579 and a ∆V of 0.00748 for a calculated Ux of
0.0447 and a calculated Ud of 0.5592. The vibrated flies showed a ∆M of 0.00114 and a ∆V of
0.001834 for a calculated Ux of 0.0007 and a Ud of 0.0082 (Table 4, Figure 3, Figure 4). The
calculated deleterious mutation rate for the vibrated flies was two orders of magnitude lower than
the control flies and is much lower than most deleterious mutation rates given in the literature
27
(Charlesworth et al. 2004, Chavarrias 2001, Drake et al. 1998, Fry 2003, Fry et al. 1999, GarciaDorado et al. 1999, Garcia-Dorado et al. 2004, Gong et al 2005, Keightly and Eyre-Walker
1999, Mukai et al. 1972, Ohnishi 1977).
Interspecies Crowding
The crowded flies exhibited a marginally significant increase in the lethal mutation rate
compared to the control. The crowded flies had eight lethal mutations in 924 chromosomes
(0.87%) scored and the control exhibited two lethal mutations in 903 chromosomes (0.27%)
scored. This gives a P = 0.063 (Table 3).
For the MA experiment the crowded flies showed a calculated ∆M of 0.01065 and a ∆V
of 0.002558 for a calculated Ux of 0.0443 and a Ud of 0.5549. The crowded flies produced a ∆M
of 0.0142 and a ∆V of 0.002344 for a calculated Ux of 0.0860 and a Ud of 1.0759 (Table 4,
Figure 5, Figure 6). This is higher than most other observed estimations of Ud in the literature
(Charlesworth et al. 2004, Chavarrias 2001, Drake et al. 1998, Fry 2003, Fry et al. 1999, GarciaDorado et al. 1999, Garcia-Dorado et al. 2004, Gong et al 2005, Keightly and Eyre-Walker
1999, Mukai et al. 1972, Ohnishi 1977).
28
DISCUSSION
Recessive Sex-Linked Lethal Mutation Rates
The data for the hypothesis that small wild-caught male flies will have a higher mutation
rate than large wild-caught male flies was inconclusive. This was an ongoing study with data
collected over a period of four summers. This data is summarized by year in Table 2. Although,
with the exception of 2004, each year saw more small fly mutations than large fly mutations, the
difference is not statistically significant for any year or for the total. This contradicts the
findings of Ivannikov and Zakharov (1996) and Ivanov and Ivannikov (1997). There may be
several reasons for this discrepancy. First, Ivannikov and Zahkarov (1996) and Ivanov and
Ivannikov (1997) used body weight instead of body length to determine the difference between
small and large male flies. It may be that this is a better indicator of flies that have been stressed
and those that have not. Secondly, we arbitrarily picked a cut-off length of 0.25mm to delineate
between small and large flies. It may be that a bigger difference in body size is needed. In the
research of Ivannikov and Zakharov (1996) and Ivanov and Ivannikov (1997) there was also an
intermediate size. This helped to truly demarcate small and large flies. It may be that we need to
breed wild-caught flies in captivity under both optimal and stressed conditions and observe any
difference in offspring size to determine what constitutes an "abnormally" small fruit fly.
We may also need to have a "gap" between large and small flies to make sure that we are
truly getting a statistically significant difference between our large and small flies. With this in
mind, all the data from the male flies of intermediate length between 0.225mm and 0.25mm were
removed. A reanalysis of the data removing all flies that fell in this "gap" generated a
comparison of four mutations in 2046 (0.20%) gametes for the large males and 15 mutations in
2695 (0.56%) gametes for the small males. This gives a P value of 0.0522, which is marginally
29
significant. A better understanding of what constitutes an "abnormally" small fly could make this
experiment more useful when comparing lethal mutation rates.
In calculating a base rate for new X-linked lethal mutation rates in D. melanogaster,
Gong et al. (2005) estimated the average mutation rate to be 0.0056 with their data ranging from
0.0042 - 0.0078 in three separate runs. Woodruff et al. (1983) compiled the results from various
studies and found a range of mutation rates for new sex-linked lethal mutations to vary from 0.0
to 0.0032. From Woodruff et al. (1983) and Gong et al (2005), we calculated an average lethal
mutation rate of 0.0016. Thus, the mutation rates calculated for the control group in all three of
our experiments fall within the parameters of many previous studies (Table 2, Table 3). The
mutation rate observed for the vibrated flies is at the higher end of the calculated rates. The
crowded flies show a rate higher than any of the cited studies. Thus, vibrational stress does seem
to have a significant effect on the rate of lethal X-linked mutations, however, the effects of
crowding on the lethal mutation rate is not as clear.
While all three experiments showed an increase in new, lethal mutations over their
respective controls, the increase in the vibrated flies was significant with a P = 0.0380 at the 95%
confidence interval, and the increase in the crowded flies, with a P = 0.0634 (or P = 0.0052 for
modified data), was marginally significant at this level. These results suggest that stress factors
of very different types (vibration vs. crowding) can increase the rate of new lethal mutations. If
confirmed, the implications of this may be important for both conservation and evolution. One
consideration may be the breeding of small populations of endangered animals under stressful
conditions in captivity, which may cause an increase in mutation rates.
Inbreeding depression is normally thought of as the decrease in fitness due to the mating
of closely related individuals. The mating of these individuals brings deleterious mutations that
30
normally present in the population (the standing gene pool) in the heterozygous state together. In
the homozygous state these deleterious alleles can be fully expressed and lower the fitness of
inbred individuals (Gaggiotti and Hanski 2004). The degree of inbreeding depression in a
population has been defined as the probability that two alleles of a gene in an individual are
identical by descent (Goodnight 2004). This assumes that inbreeding depression is dependent
upon the standing gene pool of deleterious mutations with no regard to the effect of new
mutations. The role of lethal alleles in contributing to inbreeding depression has been discussed
by more than one source (Charlesworth and Charlesworth 1987, Crow and Simmons 1983,
Halligan and Keightley 2003). Lethal alleles may contribute as much to inbreeding depression
as minor effect deleterious alleles (Halligan and Keightly 2003). Charlesworth and
Charlesworth (1987) estimated that recessive lethal alleles cause about half of the inbreeding
load in Drosophila. The role of new mutations in maintaining deleterious mutations in
populations undergoing inbreeding depression has also been discussed, with Charlesworth and
Charlesworth (1999) stating that new mutations are probably the major source for maintaining
deleterious mutations in a populations undergoing inbreeding depression. This indicates that an
increase in the rate of new recessive lethal mutations could have serious effects on the severity of
inbreeding depression and could also lead to miscalculations of the degree of inbreeding in a
population if calculations are based solely on degree of relatedness.
Many studies have linked stress with an amplification of the effects of inbreeding
depression (Gaggiotti and Hanski 2004). Keller et al. (2002) observed that in cactus finches on
the Island Daphne Major in the Galapagos, the effects of inbreeding on the population was only
observable during periods of reduced food availability or increased competition. Likewise
Frankham (2005) showed that rates of inbreeding elevated under stressful conditions using D.
31
melanogaster. What if part of this observed increase in the cost of inbreeding depression was
due not only to the amplification of already existing inbreeding depression, but also to the
increase in the mutation rate for new lethals brought about by the stressful conditions?
In a small population these new lethals may lead to extinction at a much faster rate than
estimated. The implications for conservation of endangered animals may be of particular
interest. Many animals being bred in captivity are being exposed to a wide variety of stresses,
including crowded conditions (Frankham 2005). If these stressful conditions increase the new
lethal mutation rate, this could increase inbreeding depression impacting the breeding of
endangered animals in captivity. It may be that a major factor in inbreeding depression is not
only the concentrating of preexisting deleterious alleles in the homozygous state but also the
generation of new, lethal recessive mutations that then become homozygous through inbreeding
of small populations. Animals experiencing a higher lethal mutation rate due to the stresses of
the captive environment would have an increase in inbreeding depression at a faster rate than
expected from the expression of "stock" recessive lethal and deleterious mutations.
An increase in the mutation rate due to stress may also have implications in evolution.
The generation of new species can generally be thought to occur through changes in
environmental pressures that, through natural selection, change allelic frequencies within a
population (Coyne and Orr 2004). A study by Coyne and Orr (1989) showed a relationship
between genetic distance and reproductive isolation, the major defining factor in the biological
species concept (Ridley1996). However, if stress contributes to an increase in lethal mutations
then the same stress that may be causing changes in the genetic make-up of a population
(through changing selective pressures) that will lead to speciation may also be reducing the
population as a whole. In a small population this may result in a "race" between speciation and
32
extinction. Since most speciation is thought to occur in small, isolated (allopatric) populations
(Gavrilets 2003, Mayr 1988), competition between speciation and extinction may be a very
common phenomena.
Trade-offs: Increased Lethal and Deleterious Mutations in Populations
vs. the Possibility of New Beneficial Mutations
The argument is often made that new beneficial mutations cannot have a positive effect
on evolution because they are considered to be rare compared to either lethal or deleterious
mutations in a population. Two arguments are made countering the assumption that new
beneficial mutations cannot be a force in evolution. The first argument concerns the actual
number of beneficial mutations as opposed to lethal and deleterious.
The level of beneficial mutations in a population is generally perceived as being very low
(Bell 1997). The reasoning behind this is often circular: the organism is so well adapted that
change must, of course, be deleterious. However it has been hypothesized that even if beneficial
mutations occur at a much lower rate than deleterious ones, selection will amplify the fitness of
the beneficial mutations, giving them a relatively high probability of fixation (Whitlock and
Burger 2004). To explore this hypothesis, Peck (1994) used a computer model to demonstrate
that when a single beneficial mutation arose in a large sexually reproducing population whether
the mutation was ultimately lost or fixed was not influenced by the rate of deleterious mutations.
So, perhaps the rate of beneficial mutations in a population does not need to be high for these
genetic changes to be advantageous. This, coupled with the studies cited previously showing
that the rate of beneficial mutations may not be as low as previously estimated, could mean that a
rise in mutation rate that increases the rate of new beneficial mutations might balance out the
33
detrimental effect of the rise in deleterious mutations. Hence, the increased mutation rate would
be beneficial to a population experiencing a change in environmental pressures over the shortterm.
Increased mutation rates have already been shown to be advantageous in bacterial
populations during times of stress (Hersch et al. 2004). Indeed, not only are increased mutation
rates beneficial to bacteria when adapting to new environments during times of stress (Bjedov et
al. 2003, Elena and Lenski 2003, Foster 2004, Giraud et al. 2001, Rosche and Foster 1999,
Shaver et al. 2002, Travis and Travis 2001, Wright 2004), but this "hypermutable" ability may
be selected for in the population (Hastings et al., Rosenberg et al. 1998). Studies in both yeast
(Steele and Jinks-Robertson 1991) and green algae (Goho and Bell 2000) have shown that
environmental stress can also increase the mutation rate in eukaryotes.
A recent study by Kishony and Leibler (2003) adds to the discussion that increased
mutation rates can be beneficial despite the increase in deleterious mutations by indicating that
under certain conditions stress can actually alleviate the effects of deleterious mutations. When
mutator lines of E. coli that showed a reduction in fitness on normal media were placed on media
with certain chemical stressors, or were exposed to low temperatures, the mutator strains actually
showed greater fitness than the unmutated strains. The authors hypothesize that epistatic
interactions between the deleterious mutations in the mutated lines may actually increase fitness.
However, it could also be argued that mutations that are unfavorable in one environment may
become favorable in another. The increase in mutations in the mutated lines may have allowed
for a greater chance of mutations beneficial in the new environment thus increasing the fitness of
the lines.
34
Does an increase in the mutation rate of an organism increase the number of deleterious
mutations in the genome? Of course it does. Does this mean that beneficial mutations, because
of their hypothesized low number, can never counteract the deleterious mutations? Of course not,
or evolution would never occur. However, the role of new beneficial mutations as opposed to
standing mutations in the gene pool that have become beneficial in the new environment remains
to be seen. Could an increase in the mutation rate during stressful conditions provide an
evolutionary advantage to an organism? The preceding paragraphs argue that it could. Thus,
when stressed flies exhibited a substantial increase in the genomic mutation rate, as in this study,
this increase could be an adaptation that has evolved to provide a population with a short-term
increased gene pool on which selection can act.
The High Mutation Rate in Flies Experiencing Interspecies Crowding
The calculated genomic mutation rate in the flies subjected to crowding was high (Ud =
1.07) (Table 4). A regression analysis of the slopes of both the mean and the variance are
significant (P= 0.024 and P=0.00 respectively). However, others have also reported high Ud
values. Among the three runs used to calculate Ud in D. melanogaster, Gong et al. (2005)
reported an estimated Ud = 0.68 and Ud = 0.79 for two runs. Using the confidence intervals
provided by the investigators, both of these values have an upper limit exceeding 1.0. In
addition, Fry (2001) discusses several studies done by Mukai (1972) and Ohnishi (1977) in
which a deleterious mutation rate of 1.0 is calculated for D. melanogaster. Recent studies in
Caenorhabditis elegan, which utilized direct DNA sequencing, have generated calculated
genomic mutation rates of 1.0 or higher (Denver et al. 2004, Keightly and Charlesworth 2005).
Several authors have hypothesized that high rates of mildly deleterious mutations could
have driven the evolution of sexual reproduction (Kondrashov 1988, Peck 1994). It has been
35
shown that sexual selection is better than asexual selection in ridding the genome of deleterious
mutations (Kondrashov 1988). This is because in a sexual organism deleterious mutations may
be concentrated on one chromosome through recombination and then selected against in a group
(Crow 1992). This allows many deleterious mutations to be purged from the genome at once.
Kondrashov (1988) has postulated that a Ud value greater than 1.0 for deleterious mutations in a
population may have caused the evolution of sexual reproduction due to the greater efficiency in
selecting against the new mutations. Using computer simulations Howard and Lively (1994)
showed that when a population with both sexual and monotypic asexual lineages was stressed by
coevolving parasites the rate of genomic mutation was a major determining factor in whether the
asexual population replaced the sexual or vice versa. With Ud = 0.5, asexual reproduction
becomes advantageous as long as the effect of the mutations (s) is small (s = 0.0125 or less). An
increase in s to 0.025 caused neither sexual nor asexual reproduction to have a clear advantage.
Increasing Ud to 1.0 with s = 0.125 had the same effect, ie., neither sexual nor asexual
reproduction having a clear advantage over the other. However, when Ud = 1.0 and s = 0.025
then sexual selection has a tremendous advantage. Thus, it may be reasonable to assume that
mutation rates that are increased under stress (in this case parasitism) may not only play a large
role in maintaining sexual reproduction in modern organisms but may also have played a large
role in the evolution of sexual reproduction. Evidence for high mutation rate under stress in
eukaryotes is less prevalent, but still exists. In a study of observed fitness changes in
Chlamydomonas under various stress factors, Goho and Bell (2000) hypothesized that the
observed response was due to an increase in the genome wide mutation rate and that the mutation
rate had been raised by an order of magnitude from the control. However, they did not calculate
exact mutation rates but instead based their calculations on observed difference in the means and
36
variances between control and stressed populations. In a similar study Steele and JinksRobertson (1992) showed an exponential increase in per cell mutation rate in stressed
Saccharomyces cerevisae; but this data is again hard to compare as they did not calculate a
genome wide mutation rate. So while these studies suggest a very high mutation rate under
stress for certain eukaryotic organisms, a direct comparison with our data is impossible. It may
be that Ud rates of 1.0 or greater are common in stressed populations and this high rate of
mutation not only works to maintain the widespread prevalence of sexual reproduction in
modern organisms but also played a role in the development of sexual reproduction itself. Thus,
future investigation may show that the observed Ud of 1.07 in this study is not uncommon for
stressed populations.
37
CONCLUSION
This study has provided preliminary data supporting the hypothesis that increasing the levels of
environmental stress increases the genomic wide mutation rate in D. melanogaster. If this holds
true, then these findings could have a great impact on the role of mutations in evolutionary
thought. Modulating mutation rates could explain how populations may react quickly to adapt to
new environmental pressures without succumbing to a build up of deleterious mutations. This
has already been shown to happen in prokaryotes but not in eukaryotes, where recombination
and long generation times might hinder the effectiveness of modulating mutation rates to affect
adaptation. Future work needs to be done both in confirming the observed increase in mutation
rates under stress and to try and ascertain if these increased mutation rates then decrease once the
environmental pressures have been relaxed. Once this has been confirmed the next step would
be to try and ascertain the mechanism by which these mutation rates are modulated in
eukaryotes.
38
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TABLES
53
Small females
Large females
Average wing size of
wild-caught females
2.1mm
2.72mm
Average wing size of
F1 generation females
2.72mma
2.78mma
Table 1a. Comparison of average wing length for F1 generation from the mating of wild-caught
large and small female D. melanogaster to laboratory stock males. a P = 0.355.
Small males
Large males
Average body size of
wild-caught males
1.99mm
2.52mm
Average body size of
F1 generation males
2.37mma
2.42mma
Table 1b. Comparison of average body size for F1 generation for the mating of wild-caught large
and small male D. melanogaster to laboratory stock females. aP = 0.529.
54
Year
2002
2003
2004
2005
Total
Mutation Rate
Ratio mutated/non-mutated gametes
(large flies)
1/846a
3/397 b
4/944 c
0/367 d
8/2554 e
0.0031
Ratio mutated/non-mutated gametes
(small flies)
6/1004 a
5/341 b
3/1047 c
2/674 d
16/3066 e
0.0052
Table 2. Lethal mutation rate for wild-caught male D. melanogaster.
a
P = 0.096
b
P = 0.358
c
P = 0.607
d
P = 0.0297
e
P = 0.234
55
Crowded Flies
Crowded Control
Lethal
chromosomes
8
2
Chromosomes
scored
924
903
Mutation rate
0.0087a
0.0027a
Vibrated Flies
Vibrated Control
8
2
1116
1250
0.0072b
0.0016b
Table 3. Lethal mutation rates for D. melanogaster stressed with interspecies crowding or
vibration. aP = 0.063, bP = 0.038
56
Crowded
Crowded Control
# of lines
38
55
∆M
0.01420
0.01065
∆V
0.002344
0.002558
Ux
0.0860
0.0443
Ud
1.0759
0.5549
Vibrated
Vibrated Control
52
65
0.00114
0.00579
0.001834
0.000748
0.0007
0.0447
0.0082
0.5592
Table 4. Deleterious genomic mutation rates for the X chromosome (Ux) and the diploid genome
(Ud). ∆M is the rate of decline in mean viability and ∆V is the rate in variance of viability
between lines.
57
FIGURES
58
Binscy
C(1; YS) oc ptg
females
G0
Binscy
C(1; YS) oc ptg
females
G1
Binscy
C(1; YS) oc ptg
females
G2
Binscy
C(1; YS) oc ptg
females
x
Binscy
Y
x
C(1; YS) oc ptg
RYL
males
x
C(1; YS) oc ptg
RYL
males
x
C(1; YS) oc ptg
RYL
males
males (lethal free)
Sterile progeny
Binscy/RYL males
C(1; YS) oc ptg/C(1; YS) oc ptg females
Sterile progeny
Binscy/RYL males
C(1; YS) oc ptg/C(1; YS) oc ptg females
G3, G4,… and so on…
Figure 1. The mating scheme for the accumulation of deleterious mutations in the Binscy X chromosome of Drosophila
melanogaster. In every generation (G0, G1,G2…etc), one single female and one single male are randomly selected to mate.
59
P
Single +/Y male
(from a natural population)
x
F1
Single +/Basc female
F2
+/Y males
(scored for absence, if the
chromosome determines a recessive
lethal or visible mutant phenotype)
Basc/Basc female
x
Basc/Y male
+/Basc females
(retained for
subsequent use for
maintenance stock)
Figure 2. The Basc mating scheme for the detection of X-linked lethal mutations in wild-caught
males in Drosophila melanogaster.
60
Variable
control (so lid)
v ibrated (dashed)
0.7
Mean Viability
0.6
0.5
0.4
0.3
0.2
0
2
4
6
Generations
8
10
Figure 3. Changes in mean viability (∆M) over time for deleterious mutations in D.
melanogaster experiencing vibration (Control P = 0.699, Vibrated P = 0.710).
12
61
0.09
Variable
contro l (solid)
v ibrated (dashed)
0.08
Variances
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
2
4
6
Generations
8
10
12
Figure 4. Changes in variance (∆V) over time for deleterious mutations in D. melanogaster
experiencing vibration (Control P = 0.172, Vibrated P = 0.238).
62
Variable
contro l (solid)
cro w ded (dashed)
Mean Viability
0.5
0.4
0.3
0.2
0.1
0
2
4
6
Generations
8
10
12
Figure 5. Changes in mean viability (∆M) over time for deleterious mutations in D.
melanogaster experiencing interspecies crowding (Control P = 0.336, Crowded P = 0.024).
63
Interspecific Crowding Experiment
0.14
Variable
co ntrol (so lid)
crow ded (dashed)
0.12
Variances
0.10
0.08
0.06
0.04
0.02
0
2
4
6
Generations
8
10
12
Figure 6. Changes in variance (∆V) over time for deleterious mutations in D. melanogaster
experiencing interspecies crowding (Control P = 0.367, Crowded P = 0.000).