Group Title: BMC Evolutionary Biology
Title: Environmental stress and reproduction in Drosophila melanogaster: starvation resistance, ovariole numbers and early age egg production
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Title: Environmental stress and reproduction in Drosophila melanogaster: starvation resistance, ovariole numbers and early age egg production
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Language: English
Creator: Wayne, Marta
Soundararajan, Usha
Harshman, Lawrence
Publisher: BMC Evolutionary Biology
Publication Date: 2006
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Abstract: BACKGROUND:The Y model of resource allocation predicts a tradeoff between reproduction and survival. Environmental stress could affect a tradeoff between reproduction and survival, but the physiological mechanisms underlying environmental mediation of the tradeoff are largely unknown. One example is the tradeoff between starvation resistance and early fecundity. One goal of the present study was to determine if reduced early age fecundity was indeed a robust indirect response to selection for starvation resistance, by investigation of a set of D. melanogaster starvation selected lines which had not previously been characterized for age specific egg production. Another goal of the present study was to investigate a possible relationship between ovariole number and starvation resistance. Ovariole number is correlated with maximum daily fecundity in outbred D. melanogaster. Thus, one might expect that a negative genetic correlation between starvation resistance and early fecundity would be accompanied by a decrease in ovariole number.RESULTS:Selection for early age female starvation resistance favored survival under food deprivation conditions apparently at the expense of early age egg production. The total number of eggs produced by females from selected and control lines was approximately the same for the first 26 days of life, but the timing of egg production differed such that selected females produced fewer eggs early in adult life. Females from lines selected for female starvation resistance exhibited a greater number of ovarioles than did unselected lines. Moreover, maternal starvation resulted in progeny with a greater number of ovarioles in both selected and unselected lines.CONCLUSION:Reduced early age egg production is a robust response to laboratory selection for starvation survival. Ovariole numbers increased in response to selection for female starvation resistance indicating that ovariole number does not account for reduced early age egg production. Further, ovariole number increased in a parallel response to maternal starvation, suggesting an evolutionary association between maternal environment and the reproductive system of female progeny.
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Research article


Environmental stress and reproduction in Drosophila melanogaster.
starvation resistance, ovariole numbers and early age egg
production
Marta L Waynel, Usha Soundararajan2 and Lawrence G Harshman*2


Address: 'Department of Zoology, University of Florida, Gainesville, FL32611, USA and 2School of Biological Sciences, University of Nebraska-
Lincoln, Lincoln, NE 68588, USA
Email: Marta L Wayne mlwayne@zoo.ufl.edu; Usha Soundararajan ushasoundar@hotmail.com;
Lawrence G Harshman* lharsh@unlserve.unl.edu
* Corresponding author



Published: 18 July 2006 Received: 25 July 2005
BMC Evolutionary Biology 2006, 6:57 doi: 10.1 186/1471-2148-6-57 Accepted: 18 July 2006
This article is available from: http://www.biomedcentral.com/1471-2148/6/57
2006 Wayne et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: The Y model of resource allocation predicts a tradeoff between reproduction and
survival. Environmental stress could affect a tradeoff between reproduction and survival, but the
physiological mechanisms underlying environmental mediation of the tradeoff are largely unknown.
One example is the tradeoff between starvation resistance and early fecundity. One goal of the
present study was to determine if reduced early age fecundity was indeed a robust indirect
response to selection for starvation resistance, by investigation of a set of D. melanogaster
starvation selected lines which had not previously been characterized for age specific egg
production. Another goal of the present study was to investigate a possible relationship between
ovariole number and starvation resistance. Ovariole number is correlated with maximum daily
fecundity in outbred D. melanogaster. Thus, one might expect that a negative genetic correlation
between starvation resistance and early fecundity would be accompanied by a decrease in ovariole
number.
Results: Selection for early age female starvation resistance favored survival under food
deprivation conditions apparently at the expense of early age egg production. The total number of
eggs produced by females from selected and control lines was approximately the same for the first
26 days of life, but the timing of egg production differed such that selected females produced fewer
eggs early in adult life. Females from lines selected for female starvation resistance exhibited a
greater number of ovarioles than did unselected lines. Moreover, maternal starvation resulted in
progeny with a greater number of ovarioles in both selected and unselected lines.
Conclusion: Reduced early age egg production is a robust response to laboratory selection for
starvation survival. Ovariole numbers increased in response to selection for female starvation
resistance indicating that ovariole number does not account for reduced early age egg production.
Further, ovariole number increased in a parallel response to maternal starvation, suggesting an
evolutionary association between maternal environment and the reproductive system of female
progeny.





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Background
Differential allocation of resources can give rise to trade-
offs that are manifest as negative associations between
traits [ 1,2]. Tradeoffs presumably exert a central role in life
history evolution given that energy-limited organisms
cannot simultaneously maximize all components of fit-
ness. For an insect with ephemeral food sources, such as
D. melanogaster, starvation resistance is a complex trait
which is quite likely to interact with canonical life history
traits such as fecundity [3,4]. In order to understand the
evolutionary response of this (and other) species to food
deprivation, it is important to investigate the relationship
between resistance to food deprivation and life history
traits.

Ovariole number variation is related to ecological condi-
tions. Among related species of fruit flies, a greater
number of ovarioles is correlated with "niche breadth"
measured as the number of different kinds of fruits used
by ovipositing females [5-7]. It is not clear that the fresh
fruit resource of these flies is comparable to the rotting
fruit habitat ofD. melanogaster, but the main point is that
there is an association between ovariole number and envi-
ronmental variation. Another line of evidence for the
importance of ecological variation is that D. melanogaster
ovariole number varies with latitude on three continents:
a Europe-Africa transect [8,9]; North America [10]; and
Australia [11]. Such replication of lines strongly implies
the action of selection in the creation and maintenance of
the lines, as argued for an isozyme cline [12] or quantita-
tive traits such as body size [13]. These lines in ovariole
number might be associated with differences in tempera-
ture, duration of reproductive period, the seasonal pattern
of food availability and geographic variation in over-win-
tering conditions. The evidence from both between and
within species suggests that there may be a relationship
between environmental variation and ovariole number.

Artificial selection in the laboratory is one approach to
investigate the relationship between life history and stress
resistance traits. In particular, indirect responses to artifi-
cial selection can reveal sets of correlations among traits
some of which may suggest underlying mechanisms [14-
17]. Starvation resistant lines have been shown to have a
relatively long development time [18,19], and most rele-
vant to the present study, starvation selected-lines have
been observed to be genetically correlated with relatively
reduced early age fecundity [20]. The latter association
does not prove that the traits are linked, but it does suggest
precedence for some of the trait relationships investigated
here.

In the present study, we examined the relationship
between starvation resistance, ovariole number and
fecundity in lines of D. melanogaster that were artificially


selected for resistance to female starvation. Our working
hypothesis was that females selected for starvation resist-
ance would exhibit decreased egg production at the age
they were selected, presumably as a change in allocation
from the ovary to the body, possibly by reducing ovariole
number. Starvation resistance was negatively genetically
correlated with reduced early age fecundity. However,
total egg production for the first 26 days of life was not
different between selected and control lines. Ovariole
number increased in response to selection for starvation
resistance. This increase cannot be explained by a change
in linear body size between selected and unselected lines.
To further investigate the unforeseen positiveassociation
between starvation resistance and ovariole number, we
studied the plastic and phenotypic relationships between
starvation and ovariole number. Maternal starvation
resulted in an increase in progeny ovariole number indi-
cating an integral relationship between maternal food
deprivation and the state of the ovary in the next genera-
tion.

Results
Ovariole number
Based on a comparison of progeny from unstarved moth-
ers, there were a greater number of ovarioles in selected
line females than in control line females (Figure 1; P <
0.001). Figure 1 presents the grand average number of the
sum of ovarioles from both ovaries + SE for the control
and selected lines when the latter were two generations
removed from the selection experiment regime. The
standard errors were derived from the individual line
means within line type (selected or control). The mean
sum of ovarioles per female for the five control lines was
31.80, 36.60, 32.10, 34.20, and 35.60; and the mean sum
of ovarioles per female for the five selected lines was
41.90, 39.70, 40.90, 44.40, and 38.90 (Table 4). Selected
lines have significantly more ovarioles than control lines,
whether the dams were zero, one, or two generations
removed from the selective regime (mean of control lines
vs. mean of selected lines: 34.48 vs. 42.17, 38.46 vs.
43.00, 38.39 vs. 44.15; Tables 1, 2, and 3, respectively).
Dams refers to females used to produce daughters for
ovariole number determination. Zero generations
removed from the selective regime is the case in which the
dams were taken from selected lines. One generation
removed from the selective regime refers to the case in
which the selection was relaxed for one generation in the
dams of daughters used for ovariole number determina-
tion. Two generations removed from the selection regime
refers to the case in which selection was relaxed for two
generations in the dams of daughters used for ovariole
number determination.

Maternal starvation increased the number of ovarioles in
progeny of selected and control lines females. Two gener-


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5L

0Control
Control


D Starved
E Unstarved


Selected


Figure I
Grand average of sum of ovarioles for both ovaries, for
selected (Si, right) and control (W, left) lines. The grand
average was the overall mean for the five selected and five
control lines; the standard error is based on the number of
replicate selection and control lines. The experimental treat-
ment was maternal starvation for either 28 hours or 32
hours (starved, shaded bars) compared to no maternal star-
vation (unstarved, open bars). The progeny of these females
were used for ovariole number determination. The females
("dams") subjected to the experimental treatment were two
generations removed from the selection regime as described
in the Ovariole number section of the Methods or in the
Results section. Significant differences between linetypes, but
not between line (linetypes), are the same if flies are zero,
one or two generations removed from selection.


nations removed from selection, there was a significant
effect of parental starvation resulting in increased ovariole
number (starved vs. unstarved: 42.22 vs. 40.32; treatment;
P < 0.001) as well as significant interactions between
block and treatment, and line type (selected vs. control
lines), treatment, and block (Table 3; P < 0.048 and 0.001,
respectively). A similar result was seen when parental flies
were taken directly from the selected and control lines
(Table 1). Line type (selected vs. control) was again signif-
icant (control = 34.48, selected 42.17; P < 0.001), as was
maternal starvation (starved vs. unstarved dams: 39.04 vs.


37.61; P < 0.002). There was evidence for genetic variation
within selected and control lines(line [line type]) for
ovariole number in flies whose dams were zero or one
generation removed from selection (Tables 1 and 2, line
(line type), P < 0.0001). There was no statistical support
for genetic variation within selected or control lines in
flies whose dams were two generations removed from
selection (Table 3), but this is likely more reflective of the
relatively large and significant line (line type) treatment
* block term in the denominator of the F test rather than
reflective of the biology.

Egg production
Figure 2 presents the daily line type grand average number
of eggs for selected and control line females for each of the
first 26 days of adult life. The total lifetime egg production
per female was similar for selected and control line
females (line type P = 0.1113; control, 6319.30; selected,
6037.80). There was no significant genetic variation
among lines within selected or control lines (line [line
type]; P= 0.19250). The following were the line means for
control: 6058.0, 6245.0, 6326.0, 6383.5, 6584.0 and line
means for selected: 5543.0, 5994.0, 6177.0, 6214.5,
6260.5). However, there was strong support for an effect
of day (P < 0.001) and an interaction between day and
line type (selected vs. control lines; P < 0.0001) on daily
fecundity. Motivated by the interaction, a comparison of
selected and control lines each day was made assuming an
AR(1) process. There were statistically significant differ-
ences on days 1-6 (P < 0.001), when the selected lines
were producing substantially fewer eggs than the unse-
lected lines. After day 11, the selected line females tended
to produce relatively more eggs for the remainder of the
study, but this difference was not statistically significant.

Wing length
There was no effect of selection on wing length when
selected females were one generation removed from the
selection regime (Table 4; P = 0.779). Further, there is no
relationship between ovariole number and body size,
either between selected and control lines (line type) or


Table I: Analysis of variance for sum of ovarioles from both ovaries (parents taken directly from selection regime). Comparison of
selected vs. control lines when the dams of daughters for ovariole number determination was directly from the selected and control
lines. Treatment refers to starved or unstarved mothers ("dams" as described in the Ovariole number section of the Methods or
Results). Analysis uses a two way mixed model ANOVA, with Linetype (selected vs. control) and Treatment (starved or not starved)
as fixed main effects. Linetype *Treatment is also fixed. Line refers to the five replicate lines in each linetype. Line is nested within
Linetype and is random. Line (line type) refers to variation within selected or control lines. =P < 0.05; ** =P < 0.01; *** =P < 0.001


Estimated mean squares


Variance component


Line type (selected vs. control lines)

Line type treatment
Line (line type)
Error


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Source


2956.81***
102.25**
17.41
76.54***
15.53


(fixed)
(fixed)
(fixed)
3.051
15.527


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Table 2: Analysis of variance for sum of ovarioles from both ovaries (one generation removed from selection as described in Ovariole
number section of the Methods or Results). Comparison of selected vs. control lines, using a one-way mixed model ANOVA, with line
type (control or selected) as a fixed main effect and line nested within linetype as a random effect. Line (line type) refers to the five
replicate lines in each line type. =P < 0.05; ** =P < 0.01; *** =P < 0.001


Source


Line type (selected vs. control lines)
Line (line type)
Error


Estimated mean squares

1027.56*
133.17***
28.04


Variance component

(fixed)
5.28
28.04


within each type of line (line [line type]). Across line
types, the correlation between ovariole number and body
size is not significant (R = 0.09776; P = 0.7882). Within
line types, there is also no relationship between the two
traits (control: R = -0.6385, P = 0.2462; selected: R =
0.6001, P = 0.2846). Results from non-parametric analy-
ses are consistent with parametric analyses (data not
shown).

Starvation resistance
To assess the direct response to selection at the end of the
present study, starvation resistance was assessed in the
same manner used in the selection experiment except that
starvation continued until all flies died. The grand mean
female survival time under starvation was 98 (+/- 10)
hours for the selected lines and 62 (+/- 15) hours for the
control lines. These mean values are similar to those for
selected and control line females at generation 27 [21]
which was five generations before the present study
began. Thus, the marked difference in starvation resist-
ance between selected and control line females, was main-
tained throughout the present study.


Discussion
Selection for increased adult female starvation resistance
resulted in a correlated increase in the number of ovari-
oles. Similarly, maternal starvation resulted in an increase
in offspring ovariole number. The present study was con-
sistent in terms of an increase in ovariole number in
selected females, irrespective of the assay generation or of
the number of generations removed from the selection
regime. Moreover, maternal starvation consistently
resulted in an increase in progeny ovariole number in
both control and selected line females. One might expect
the effect of maternal starvation on ovariole number to be
substantially greater in control females because 28 32
hours of starvation would be expected to have greater
physiological impact on these females than on selected
females. On the other hand, selected line females might
have evolved to become more responsive to starvation
conditions. Our study provides evidence for a consistent
effect of starvation on ovariole number, which should
motivate additional research on underlying mechanisms
including the effect of duration of maternal starvation in
the context of evolved starvation resistance.


The lines used for the present study were produced by
selection only on female starvation resistance [22]


Table 3: Analysis of variance for sum of ovarioles from both ovaries (two generations removed from selection as described in Ovariole
number section of the Methods or Results). Comparison of selected vs. control lines via a 3-way, mixed model ANOVA. Linetype
(selected vs. control lines), treatment (starved or not starved), block (28 hours or 32 hours starvation) are fixed main effects. Their
interactions are also fixed. Line refers to the five replicate lines in each linetype; line is nested within linetype and is random, as are the
interactions of line with block and treatment. =P < 0.05; ** =P < 0.01; *** =P < 0.001


Source


Line type (selected vs. control
lines)
Treatment
Block
Line type treatment
Line type block
Treatment block
Line type treatment block
Line (line type)
Line (line type) treatment
Line (line type) block
Line (line type) treatment block
Error


Estimated mean squares

4965.13***


545.3 ***
1.71
47.04
220.83
291.21*
7.26
191.32
18.58
80.83
53.29**
20.99


Variance component

(fixed)

(fixed)
(fixed)
(fixed)
(fixed)
(fixed)
(fixed)
2.420
0.000
0.918
2.153
20.992


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400-

350--

300--


200-

150-

100-


50 +


Control
- ---- Selected


1 5 10 15 20 25


Day


Figure 2
The daily fecundity schedule for the selected and control lines. The grand average per day was the overall mean number of eggs
for selected lines and control lines per bottle with ten females in each bottle.


because of the heterogeneity observed among starvation-
selected lines when females and males were selected [19].
Both female and male starvation resistance evolved in
response to selection on females [22]. Indirect male
responses to selection have not been investigated in lines
selected for female starvation resistance [22]. It is not clear
what the likely correlated responses in males would be,
but it might be the case that male reproductive perform-
ance paralleled the pattern of female egg production,


showing a relative decrease at early ages in selected lines
(Figure 2). Selection only on females might have contrib-
uted to the pattern of indirect responses in the present
study. However, selection only on females probably does
not explain the relationship between starvation resistance
and reduced early age fecundity because a decrease in
early age fecundity was previously observed to be a corre-
late of selection for starvation resistance when both sexes
were employed in the selection regime [20].


Table 4: Ovariole number (sum of ovarioles from both ovaries) and wing length of daughters from unstarved mothers two generations
removed from selection (as described in Ovariole number section of the Methods or Results). Selection for female starvation resistance
did not result in an increase in linear body size, and linear body size is not correlated with ovariole number within lines.


Mean ovariole number std. error Mean wing measurement (mm)
std. error


1.607 0.008
1.654 0.008
1.661 0.010
1.669 0.008
1.669 0.007
1.615 0.008
1.628 0.009
1.698 0.008
1.667 0.009
1.682 0.007


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Line number


Line type


control
control
control
control
control
selected
selected
selected
selected
selected


31.80 1.20
36.60 1.64
32.10 1.27
34.20 2.13
35.60 1.32
41.90 0.85
39.70 0.58
40.90 1.22
44.40 1.17
38.90 1.16


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The indirect responses to selection, increased ovariole
number and decreased early age fecundity, were not a
result of cryptic selection for an increase in linear body
size, because there was no difference in wing length
between selected and control lines. Wing length was cho-
sen as an estimate of body size in preference to estimates
of mass for two reasons: first, because differences in ovari-
ole number could contribute to differences in mass; and
second, because it is well established that laboratory selec-
tion for starvation resistance results in an accumulation of
lipid in flies [18,19] and hence an increase in weight.
Body size as estimated by wing size or thorax length is
often correlated with development time [23-25]. Gener-
ally, slower development time was correlated with greater
thorax or wing length and vice versa, but this relationship
was not found in all studies. In any case, if there was any
difference in development time between selected and con-
trol lines in the present study, then it was not manifest as
a difference in a linear measure of body size (wing
length).

The response to selection in the present study might con-
form to the Y model of resource allocation in which
endogenous resources are allocated to either survival or
reproduction [26]. When lines were selected for starvation
resistance early in life, female flies responded by reducing
early age reproduction in favor of survival under starva-
tion conditions. Although the indirect responses of simi-
lar laboratory selection experiments using Drosophila are
sometimes heterogeneous [3,27], our results were consist-
ent with another selection experiments in which starva-
tion resistance was genetically correlated with reduced
early age fecundity [20]. Thus, in both studies it is plausi-
ble that the females selected for early age starvation resist-
ance have shifted energy away from reproduction to
survival under starvation conditions.

Energetic studies on selected and control lines of D. mela-
nogaster are relevant to the relationship between reproduc-
tion and starvation resistance. Energy storage compounds
have been measured in lines selected for starvation resist-
ance and there is an increase in lipid and glycogen content
[19-21,28,29]. Reduced reproduction does not arise from
relatively low metabolic rate in the selected lines; there is
little evidence that metabolic rate decreases in lines
selected for starvation resistance [22,30]. One might
expect a tradeoff between somatic energy storage and
reduced energy in the reproductive system for egg produc-
tion, but is not clear that energy for reproduction is limit-
ing in the laboratory environment [31].

An alternative to an energetic explanation for the observa-
tions in the present study is derived from consideration of
the role of molecular signaling in life history evolution
[32]. The basic argument is that the ovary could be a


source of signals that affect the soma independent of the
role of the ovary as a sink for nutrients used for reproduc-
tion. As reviewed in Leroi (2001), [32], there is evidence
for such signals in Caenorhabditis elegans and these signals
require a functional insulin signaling pathway. The D.
melanogaster insulin signaling pathway is conserved in
that it shares common features with pathways in C. elegans
and mammals [33]. The insulin signaling pathway could
play a role in biochemical and life history lines docu-
mented for D. melanogaster [13]. However, a homozygous
null mutation in the D. melanogaster insulin receptor sub-
strate protein gene, which blocks normal insulin signal-
ing, has little effect on ovariole number in an outbred wild
type genetic background [34]. Nevertheless, signaling
from the ovary could act as a mediator of egg production
as an indirect response to selection for starvation resist-
ance.

Greater numbers of ovarioles in the selected lines were
correlated with reduced early age fecundity, but there was
no correlation with overall fecundity. Earlier studies
found a negative genetic correlation between ovariole
number and early age reproductive maturation in a selec-
tion experiment for longevity and late age reproduction
although there was no clear association between late life
ovariole number and altered vitellogenic egg maturation
[35,36]. Does the early age negative correlation imply that
that there is a tradeoff between ovariole number and early
age fecundity? Perhaps, but it will be necessary to estab-
lish a functional relationship between the traits to rigor-
ously document whether a tradeoff exists [37]. Such a
tradeoff may be relatively pronounced in the context of
starvation resistance. Many studies of unperturbed natural
populations have found a positive association between
ovariole number and early fecundity [38-40]. Candidate
genes associated with starvation resistance tend to be in
developmental pathways responsible for cell fate [41].
These genes might exert pleiotropic effects during the pre-
adult stages when ovarioles develop, whereby alleles con-
ferring starvation resistance could cause an increase in
ovariole number. Interestingly, an increase in the number
of ovarioles would be expected to increase the total
number of oocyte stem cells per ovary; and that could pro-
mote greater fecundity later in life assuming that a decline
in stem cell number and function underlies the decline in
late-age reproductive potential [42], as well as the lack of
difference in total 26 day fecundity between selected and
control lines in this study, despite the decrease in early
fecundity in the selected lines. This could also explain the
positive genetic correlation between longevity and ovari-
ole number in lines selected for late life reproduction and
extended longevity [36].

There is an alternate interpretation of the phenotypic
manipulation experiments reported in the present study.


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In the absence of a suitable egg laying substrate during the
time females were held in empty bottles for the starvation
treatment, fertilized eggs might develop within the
females. If such internal development resulted in a head
start for these eggs; and if the first eggs laid after 28-32
hours of starvation comprised a large proportion of the
progeny used for ovariole measurements, then it is possi-
ble that the relative maturity of these eggs could contrib-
ute to an increase in ovariole number. However, such
developmental processes could not explain the increase in
ovariole number in selected lines relative to control lines,
because the difference in ovariole number persisted even
in control lines two generations removed from the selec-
tive regime; and thus egg retention and accompanying
accelerated maturation due to lack of oviposition sub-
strates did not occur.

The dines in ovariole number within D. melanogaster [8-
11] suggest that there is a relationship between a temper-
ate environment and increased ovariole number. How-
ever ovariole number and starvation resistance are not
known to positively covary in natural populations; a shal-
low dine in starvation resistance among Australian popu-
lations [43] appears to be negatively correlated with an
Australian dine in ovariole number [11 ].

In general, there is a negative relationship between early
age fecundity and stress resistance in selection experi-
ments [37]. Moreover, phenotypic manipulation experi-
ments have documented a negative relationship between
egg production and starvation resistance [44,45]. Simi-
larly, increased stress resistance, reduced early age repro-
duction, retarded vitellogenic oocyte maturation and
increased ovariole number were all indirect responses to
selection for late life reproduction and longevity
[35,36,46-48]. In the present study, a greater number of
ovarioles was associated with reduced early age fecundity
and resistance to a specific stressor, starvation. A genetic
correlation between female starvation resistance and
increased ovariole number, and phenotypic correlation
between maternal starvation and increased ovariole
number, is documented for the first time in the present
study. In the present study, reduced early age egg produc-
tion is associated with increased ovariole number, as is
relatively high later age egg production; but it is not clear
that the larger number of ovarioles was necessary for the
relatively elevated later age egg production in the selected
lines. As a possible underlying mechanism for the rela-
tionship between ovariole number and egg production in
the present study, the ovary might be able to modulate the
distribution and allocation of endogenous energy storage
compounds in relationship to life history traits. As sum-
marized in Chippindale et al. (1993), [44], various studies
have implicated the ovary and the neuroendocrine system
as controlling factors in cellular lipid content and energy


storage compound distribution in the body. In terms of
life history traits and physiological tradeoffs, the number
of ovarioles might correspond to the physiological impact
of the ovary as a mediator of energy storage compound
allocation between reproduction and the soma. Whether
caused by energy compound shifts in the adult or pre-
adult developmental pathways, the parallel phenotypic
and genetic association between starvation resistance and
ovariole number warrants further investigation.

Conclusion
Selection for female starvation resistance resulted in an
increase in ovariole number as a correlated response to
selection as well as a decrease in early age fecundity, but
total egg production was not affected by selection. Selec-
tion was associated with relatively low early age egg pro-
duction; there was a negative correlation between
starvation resistance and early age egg production and a
negative correlation between ovariole number and early
age egg production. Phenotypic manipulation by starva-
tion resistance resulted in an increase in ovariole number
in progeny. Starvation and ovariole number in progeny
are apparently genetically and phenotypically linked, but
the functional basis for this association is not yet under-
stood.

Methods
Culture conditions
The standard rearing condition for all of the present study
includes control of larval density by counting eggs, rearing
at 21 C under constant illumination and holding of
adults at 21C. These conditions were used throughout
the present study. For each experiment, assay, or morpho-
logical measurement, all selection and control lines were
simultaneously evaluated. Standard half-pint bottles and
10 dram vials were used in all experiments and a standard
cornmeal, molasses, yeast medium was used to culture the
flies.

Artificial selection
The present study was conducted from generations 32 to
40 of laboratory selection for female starvation resistance.
The details of selection are described in Harshman and
Schmid (1998), [22], but they will be summarized here.
After approximately two years of laboratory culture, a base
stock ofD. melanogaster was subdivided into 10 lines. Five
lines were selected for female starvation resistance
(referred to as the Si lines) and five lines were maintained
as controls (referred to as the W lines). When flies were 3-
4 days post-eclosion, which allows time for sexual matu-
ration and mating; females were separated from males by
light ether anesthesia and allowed to recover overnight on
food. Females were placed in empty bottles with a water-
saturated plug at a standard density. After 24 hours of star-
vation conditions, before significant mortality occurred,


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females from the control lines were transferred to fresh
vials with Drosophila food and added live yeast. The
selected line females continued to be held without food
until approximately 50% mortality and the survivors were
transferred to fresh vials with Drosophila food and added
live yeast. In general, there was no, or a few percent, mor-
tality when control females were exposed to starvation for
24 hours and typically 40 60% mortality (50% on aver-
age) across generations, among selected line females in
the selection regime. Following the regime established at
the beginning of the selection experiment [22], after 3
days in vials following sublethal starvation (control lines)
or starvation selection (selected lines), all females were
transferred to fresh Drosophila food with added yeast in
bottles at similar adult density for egg collection. Larval
density was controlled by transfer of approximately 75
eggs to each vial used to produce the next generation.
Control of larval density by counting eggs was employed
throughout the selection experiment and for all experi-
ments in the present study.

Ovariole number
The method for ovariole number determination was
described in Carlson et al. (1998 [35]). Females for dissec-
tion were reared from multiple vials and randomized
across vials. Randomized adult females were held for 5-7
days after eclosion with an equal number of males in vials
at 21 C prior to being frozen. Females were frozen and
held at -20 C until they were dissected for ovariole
number determination. Typically, ovariole numbers were
determined from these females within 30 days after they
were frozen. Only a few females were thawed at any one
time (typically 5 or less) to avoid tissue deterioration after
the freeze-thaw process. Each female was placed in a drop
of Drosophila Ringers solution (7.5 g NaCl, 0.35 g KC1.
0.21 g CaCl2 per liter of H20) for dissection. The tip of the
abdomen was pulled away in a posterior direction while
the remainder of the body was held by a second pair of
forceps. The ovarioles were gently separated from each
other using tungsten needles. All ovarioles in both ovaries
were counted in each female.

Three similar experiments were conducted on ovariole
number. In all three experiments, ovariole numbers were
determined in females from all selected and control lines.
In two of the studies, a phenotypic perturbation (maternal
starvation) was included in the comparison of selected
and control line females. Studies were conducted on flies
zero, one, or two generations removed from selection. The
motivation for using flies variable numbers of generations
removed from selection was to be able to determine if the
response to the starvation as a phenotypic manipulation,
or the magnitude of the ovary number indirect response
to selection, was related to generational proximity to
lengthy starvation as the selective agent in the selection


experiment. Moreover, it provided for a high degree of
repetition of the tests of relationship between starvation
selection, maternal starvation and ovariole number.

For zero generations removed from selection, females
used as dams in the experiments were taken directly from
the starvation lines without intervening generations of
rearing under control line conditions. Selected and con-
trol line parental females were either exposed to 28 hours
of starvation, or not starved, prior to egg collection for the
next generation which provided the daughters used for
ovariole number determinations. For this experiment,
ovarioles from both ovaries were counted from 10
females per line. For one generation removed from selec-
tion, the dams of the daughters for ovariole number deter-
mination were taken from lines in which selection had
been relaxed for one generation. All of the ovarioles from
both ovaries were counted from 20 females for each line.
For two generations removed from selection, the dams of
the daughters for ovariole number determination were
taken from lines in which selection had been relaxed for
two generations. This experiment included a comparison
of ovariole number after maternal starvation treatment
versus no starvation; half of the females from each
selected and control line were starved and half were held
without starvation. At the beginning of the experiment,
females and males were held at 21 0C for 5-7 days. There-
after, females were separated from males and held at 21 C
at a density of 10 per empty vial with a water-saturated
cotton plug for 28 hours for parental starvation in one
case and 32 hours of starvation in a second case. The
longer period of time without food, 32 hours, was nearly
the maximum duration of starvation without significant
mortality. The motivation for using the longest possible
sub-lethal starvation period in one experiment was to
investigate the maximal physiological impact of maternal
starvation on offspring ovariole number. After the starva-
tion period, all females were transferred to fresh food for
egg collection. As always, a standard number of eggs (75
per vial) from starved and unstarved, selected and control
line females was collected and transferred to each vial
used to produce offspring to be assayed. After eclosion,
males and females from the next generation were held
together on fresh food for 5-7 days. After this time
females were frozen for ovariole number determination.
Ovariole numbers were counted for both ovaries from 15
females per line. Analysis was performed on the sum of
the ovarioles from both ovaries.

Early age egg production
The focus of this assay was to determine the pattern of
early age egg production. Flies from the lines selected for
female starvation resistance, and control lines, were used
to assess daily egg production for 26 days post-eclosion.
Two generations of controlled density culture at 21C


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were conducted without starvation prior to collecting flies
for the egg production study.

The egg production assays employed cut bottles with Dro-
sophila food. The bottles were cut completely through
around the circumference approximately 2.5 cm above
the bottom of each bottle. During the process of preparing
Drosophila food, molten medium was poured almost up to
the cut that separates the bottom and top half of the bot-
tles. When the food cooled, the top of the bottle was
attached to the lower portion of the bottle with masking
tape. Two bottles were used for each of the 10 lines (5 star-
vation selected lines and 5 control lines). Each bottle con-
tained 10 females and 10 males, and the flies were 4 days
old at the beginning of the study. Every day for 26 days,
the flies in the bottles were transferred to new cut bottles
with Drosophila medium. The previous day's bottles were
disassembled and the disk of medium in the lower por-
tion of the bottle was removed and placed on a piece of
wax paper (the egged surface side up) in a box. The Dro-
sophila food disks, which contained eggs produce during a
24 hour period, were frozen at -20 0C to be thawed later
and eggs counted.

Wing length
Wings were measured to appraise whether fecundity and
ovariole number differences between selected and control
lines was correlated with changes in linear body size. The
relevance to the present study is that linear body size has
often been found to be correlated with fecundity. A
genetic correlation between ovariole number and body
size was not found in an earlier study [49], but that did
not preclude an increase in linear body size as a response
to selection for starvation resistance. A genetic correlation
between ovariole number and starvation resistance in our
study could have been caused by a difference in linear
body size between selected and control line females.

The measurements were taken under 20x magnification
using an ocular micrometer. Wing length was measured as
the linear distance from the intersection of the anterior
crossvein to the wing margin at the distal end of the third
longitudinal vein. Both wings were removed from each
female on day 4 post eclosion and the wings were posi-
tioned under a coverslip on a glass slide. Twenty pairs of
wings were measured for each selected and control line.

Starvation resistance
At the end of the selection experiment, starvation resist-
ance was determined for selected and control lines. At
generation 40, selection was relaxed for two generations
before starvation resistance was determined in the
selected and control lines before the selection experiment
was terminated. Starvation resistance was determined
using replicate bottles for each selected and control line.


For starvation assays, females were placed in empty bot-
tles with water-saturated plugs. This assay allowed for
comparison to the direct response to selection in these
lines assessed in an earlier generation [21].

Statistical analysis
The general form of the statistical analysis used in the
present study was to test for grand mean line type
(selected and control lines) differences, and treatment dif-
ferences, using an error term that included the variation
between replicate lines of the same type. Ovariole number
and wing length were statistically analyzed by ANOVA
using the GLM procedure of the SAS package, and variance
components estimated using PROC VARCOMP.

Daily egg production for 26 days was analyzed as repeated
measures. Specifically, the autoregressive 1 process,
AR(1), provided a better fit to the data than other covari-
ance structures tested including compound symmetry.
The data were analyzed as a hierarchical ANOVA using an
error term that included the variation between replicate
lines of the same type (two replicate bottles were set up for
each line). The MIXED procedure of the SAS package was
used for the analysis. The data was analyzed untrans-
formed or square root transformed; the results of the sta-
tistical analyses were essentially the same and only the
analysis of the square root transformed data is reported in
the present study.

Abbreviations
ANOVA = analysis of variance, SE = standard error, GLM
= general linear model, df = degrees of freedom

Authors' contributions
MW and LH conceived of the project. MW conducted
most of the statistical analyses, made

major contributions to writing the manuscript and revi-
sions, and figures. US collected the data in the LH labora-
tory. US entered the data into spreadsheets and provided
summaries of the data for figures. US generated a rough
draft of one figure. LH designed the experiments, wrote
the first draft of the manuscript and made major contribu-
tions to manuscript revisions.

Acknowledgements
We thank C. Gunnels IV, L. Higgins, S. Schaack, J. Stamps and Tony Zera
for comments on the manuscript. The authors also express appreciation to
David Marx (Department of Statistics, University of Nebraska-Lincoln) for
contributions to the statistical analysis. M.L. Wayne was supported by a
grant from the National Institute of Health (RO I GM59884-02). L.G. Harsh-
man was supported by the NSF EPSCoR Metabolite Signaling Center grant
(EPS-0346476) at the University of Nebraska-Lincoln.





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References
I. de Jong G, van Noordwijk AJ: Acquisition and allocation of
resources: genetic (co)variances, selection and life histories.
American Naturalist 1992, 139:749-770.
2. Stearns SC: The Evolution of Life Histories Ist edition. Oxford Univer-
sity Press; 1992.
3. Harshman LG, Hoffmann AA: Laboratory selection experiments
using Drosophila: what do they really tell us? TREE 2000,
15:32-36.
4. Parsons PA: Ecobehavioral genetics habitats and colonists.
Annual Review of Ecology and Systematics 1983, 14:35-55.
5. Fitt GP: Variation in ovariole number and egg size ofspecies
of Dacus (Diptera; Tephritidae) and their relation to host
specialization. Ecological Entomology 1990, 15:255-264.
6. Kambysellis MP, Heed WB: Studies of oogenesis in natural pop-
ulations of Drosophilidae. I. Relation of ovarian development
and ecological habitats of the Hawaiian species. American Nat-
uralist 1971, 105:31-49.
7. R'kha S, Moreteau B, Coyne JA, David JR: Evolution of a lesser fit-
ness trait: egg production in the specialist Drosophila sechel-
lia Genetical Research 1997, 69:17-23.
8. DelpuechJ-M, Moreteau B, Chiche J, Pla E, VouidibioJ, David JR: Phe-
notypic plasticity and reaction norms in temperate and trop-
ical populations of Drosophila melanogaster: ovarian size and
developmental temperature. Evolution 1995, 49:670-675.
9. Lemeunier F, David JR, Tsacas L, Ashburner M: Geographic varia-
tions and climatic adaptations. In The Genetics and Biology of Dro-
sophila Volume 3e. Edited by: Ashburner M. London: Academic Press;
1986:147-256.
10. Capy P, Pla E, David JR: Phenotypic and genetic variability of
morphometrical traits in natural populations of Drosophila
melanogaster and D. simulans. I. Geographic variations. Genet
Sel Evol 1993, 25:517-536.
I I. Azevedo RBR, French V, Partridge L: Thermal evolution of egg
size in Drosophila melanogaster. Evolution 1996, 50:2338-2345.
12. Berry A, Kreitman M: Molecular analysis of an allozyme dine:
Alcohol dehydrogenase in Drosophila melanogaster on the
east coast of North America. Genetics 1993, 134:869-893.
13. de Jong G, Bochdanovits Z: Latitudinal lines in Drosophila mel-
anogaster. body size, allozyme frequencies, inversion fre-
quencies, and the insulin-signalling pathway. journal of Genetics
2003, 82:207-223.
14. Rose MR, Graves JL, Hutchinson EW: The use of selection to
probe patterns of pleiotropy in fitness characters. In Insect Life
Cycles Edited by: Gilbert F. New York: Springer Verlag; 1990:29-42.
15. Rose MR, Nusbaum TJ, Chippindale AK: Laboratory evolution:
the experimental wonderland and the Cheshire Cat Syn-
drome. In Adaptation Edited by: Rose MR, Lauder GV. San Diego:
Academic Press; 1996:221-242.
16. Huey RB, KingsolverJG: Evolution of resistance to high temper-
ature in ectotherms. American Naturalist 1993, 142:521-546.
17. Gibbs AG: Laboratory selection for the comparative physiol-
ogist. journal of Experimental Biology 1999, 202:2709-2718.
18. Harshman LG, Hoffmann AA, Clark AG: Selection for starvation
resistance in Drosophila melanogaster. physiological corre-
lates, enzyme activities and multiple stress responses. j Evol
Biol 1999, 12:370-379.
19. Chippindale AK, Chu TJF, Rose MR: Complex trade-offs and the
evolution of starvation resistance in Drosophila melanogaster
Evolution 1996, 50:753-766.
20. Rose MR, Vu LN, Park SU, Graves JL: Selection on stress resist-
ance increases longevity in Drosophila melanogaster Experi-
mental Gerontology 1992, 27:241-250.
21. Harshman LG, Moore KM, Sty MA, Magwire MM: Stress resistance
and longevity in selected lines of Drosophila melanogaster.
Neurobiol Aging 1999, 20:521-529.
22. Harshman LG, Schmid JL: Evolution of starvation resistance in
Drosophila melanogaster. Aspects of metabolism and coun-
ter-impact selection. Evolution 1998, 52:1679-1685.
23. Cortese MD, Norry FM, Piccinali R, Hasson E: Direct and corre-
lated responses to artificial selection on development time
and wing length in Drosophila buzzatti Evolution 2002,
56:2541-2547.
24. Partridge L, Fowler K: Responses and correlated responses to
artificial selection on thorax length in Drosophila mela-
nogaster. Evolution 1993, 47:213-226.


25. Robertson FW: The ecological genetics of growth in Dro-
sophila. Genet Res 1963,4:74-92.
26. van Noordwijk AJ, de Jong G: Acquisition and allocation of
resources: their influence on variation in life-history tactics.
American Naturalist 1996, 1 28:137-142.
27. Tower J: Aging mechanisms in fruit flies. BioEssays 1996,
18:799-807.
28. Graves JL, Toolson EC, Jeong C, Vu LN, Rose MR: Desiccation,
flight, glycogen, and postponed senescence in Drosophila
melanogaster. Physiological Zoology 1992, 65:268-286.
29. Djawdan M, Chippindale AK, Rose MR, Bradley TJ: Metabolic
reserves and evolved stress resistance in Drosophila mela-
nogaster. Physiological Zoology 1998, 71:584-594.
30. Djawdan M, Rose MR, Bradley TJ: Does selection for stress resist-
ance lower metabolic rates? Ecology 1997, 78:828-837.
31. Rose MR, Bradley TJ: Evolutionary physiology of the cost of
reproduction. Oikos 1998, 83:443-451.
32. Leroi AM: Molecular signals versus the Loi de Balancement .
Trends in Ecology and Evolution 2001, 16:24-29.
33. Garofalo RS: Genetic analysis of insulin signaling in Drosophila
Trends in Endocrinology and Metabolism 2002, 13:156-162.
34. Richard DS, Rybczynski R, Wilson TG, Wang Y, Wayne ML, Zhou Y,
Partridge L, Harshman LG: Insulin signaling is necessary for vitel-
logenesis in Drosophila melanogaster independent of the roles
of juvenile hormone and ecdysteroids: Female sterility of the
chicoi insulin signaling mutation is autonomous to the ovary.
journal of Insect Physiology 2005, 51:455-464.
35. Carlson KA, Nusbaum TJ, Rose MR, Harshman LG: Oocyte matu-
ration and ovariole number in lines of Drosophila mela-
nogaster selected for postponed senescence. Functional Ecology
1998, 12:514-520.
36. Carlson KA, Harshman LG: Extended longevity lines of Dro-
sophila melanogaster. characterization of oocyte stages and
ovariole numbers as a function of age and diet. j Gerontol Ser
A-Biol Sci Med Sci 1999, 54:B432-B440.
37. Zera AJ, Harshman LG: The physiology of life history trade-offs
in animals. Annual Review of Ecology and Systematics 2001.
38. Bouletreau-Merle J, Allemand R, Cohet Y, David JR: Reproductive
strategy in Drosophila melanogaster: Significance of a genetic
divergence between temperate and tropical populations.
Oecologia 1982, 53:323-329.
39. Cohet Y, David J: Control of the adult reproductive potential
by preimaginal thermal conditions. Oecologia 1978, 36:295-306.
40. David JR: Le nombre d'ovarioles chez la Drosophila: Relation
avec la fecondite et valeur adaptive. Archives de Zoologie Exp6ri-
mentale et G6n6rale 1970, I I 1:357-370.
41. Harbison ST, Yamamoto AH, Fanara JJ, Norga KK, Mackay TFC:
Quantitative trait loci affecting starvation resistance in Dro-
sophila melanogaster. Genetics 2004, 166:1807-1823.
42. Xie T, Spradling AC: A niche maintaining germ line stemcells
in the Drosophila ovary. Science 2000, 290:328-330.
43. Hoffmann AA, Hallas R, Sinclair C, Mitrovski P: Levels of variation
in stress resistance in Drosophila among strains, local popu-
lations, and geographic regions: Patterns for desiccation,
starvation, cold resistance, and associated traits. Evolution
2001, 55:1621-1630.
44. Chippindale AK, Leroi AM, Kim SB, Rose MR: Phenotypic plastic-
ity and selection in Drosophila life-history evolution. I. Nutri-
tion and the cost of reproduction. journal of Evolutionary Biology
1993, 6:171-193.
45. Salmon AB, Marx DB, Harshman LG: A cost of reproduction in
Drosophila melanogaster. Stress susceptibility. Evolution 2001,
55:1600-1608.
46. Rose MR: Laboratory evolution of postponed senescence in
Drosophila melanogaster Evolution 1984, 38:1004-1010.
47. Service PM: Physiological mechanisms of increased stress
resistance in Drosophila melanogaster selected for postponed
senescence. Physiol Zool 1987, 60:321-326.
48. Service PM, Hutchinson EW, Mackinley MD, Rose MR: Resistance
to environmental stress in Drosophila melanogaster selected
for postponed senescence. Evolution 1985, 42:708-716.
49. Wayne ML, Hackett JB, Mackay TFC: Quantitative genetics of
ovariole number in Drosophila melanogaster. I. Segregating
variation for chromosome 3 and fitness. Evolution 1997,
51:1156-1163.




Page 10 of 10
(page number not for citation purposes)


BMC Evolutionary Biology 2006, 6:57




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