doi: 10.1111/j.1420-9101.2012.02502.x

Effects of parental radiation exposure on developmental
instability in grasshoppers
D. E. BEASLEY*, A. BONISOLI-ALQUATI*, S. M. WELCH*, A. P. MØLLER & T. A. MOUSSEAU**Department of Biological Sciences, University of South Carolina, Columbia, SC, USA
Laboratoire Ecologie, Systematique & Evolution, CNRS UMR 8079, University of Paris 11, Orsay, France

Keywords:

Abstract

environmental stress;
fluctuating asymmetry;grasshoppers;
life history;
radioactive contamination;transgenerational genomic instability.

Mutagenic and epigenetic effects of environmental stressors and theirtransgenerational consequences are of interest to evolutionary biologists
because they can amplify natural genetic variation. We studied the effect ofparental exposure to radioactive contamination on offspring development inlesser marsh grasshopper Chorthippus albomarginatus. We used a geometricmorphometric approach to measure fluctuating asymmetry (FA), wing shape
and wing size. We measured time to sexual maturity to check whether
parental exposure to radiation influenced offspring developmental trajectory
and tested effects of radiation on hatching success and parental fecundity.
Wings were larger in early maturing individuals born to parents from highradiation sites compared to early maturing individuals from low radiation sites.
As time to sexual maturity increased, wing size decreased but more sharply inindividuals from high radiation sites. Radiation exposure did not significantly
affect FA or shape in wings nor did it significantly affect hatching success andfecundity. Overall, parental radiation exposure can adversely affect offspringdevelopment and fitness depending on developmental trajectories although
the cause of this effect remains unclear. We suggest more direct measures offitness and the inclusion of replication in future studies to help further our
understanding of the relationship between developmental instability, fitness
and environmental stress.

Introduction
Genomic instability in the form of chromosomal irregularities or genetic mutations can occur because ofenvironmental stressors such as viruses (Wu et al.,2010), plant toxins (Eizirik & Kisby, 1995), mutagenic
chemicals (Mishra et al., 2009) and radiation exposure(Dubrova, 2003). This instability occurs in mitoticallydividing cells and interferes with the function of tissues
and organs (Wong et al., 2003). Transgenerational genomic instability results from genomic instability transferred from the parental germline to the somatic cells ofoffspring, leading to the destabilization of developmental
pathways (Natarajan, 2006). Exposure to external stressors and transgenerational genomic instability, which
increases sensitivity of the progeny genome to mutagens
Correspondence: D. E. Beasley, Department of Biological Sciences,
University of South Carolina, Columbia, SC 29208, USA.
Tel.: +803 777 8241; fax: +803 777 4002; e-mail: beasleyd@email.sc.edu(Hoyes et al., 2001), can have developmental and fitness
consequences for individuals inhabiting stressful environments.
Exposure to radiation was one of the first external
factors observed to induce transgenerational genomic
instability in mammalian models (Sinclair, 1964;
Nomura, 1982; Lyon & Renshaw, 1988; Luke et al., 1997;
Morgan, 1999). Additionally, transgenerational effects
from radiation exposure have also been demonstrated in
insects, particularly in the form of inherited sterilization(North & Holt, 1968, 1969; Bloem et al., 1999; Carpenter
et al., 2009). Ionizing radiation generates reactive oxygen
species (ROS) either by the oxidation of water orsecondarily by producing reactive intermediates (Rugoet al., 2002). Accumulation of ROS can result in oxidative
stress, defined as the imbalance between ROS generationand the antioxidant defences that neutralize them(Monaghan et al., 2009), which can cause an increasein mutation rates (Riley, 1994). More recently, radioactivity has been linked to inhibition of standard epigeneticª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

1149

1150

D. E. BEASLEY ET AL.

processes such as methylation (Koturbash et al., 2006),
histone modification (Rogakou et al., 1998) and RNAassociated silencing (Jaenisch & Bird, 2003), which may
result in genomic instability (Kovalchuk & Baulch,2008). These mutagenic and epigenetic effects of ionizing
radiation and their consequences across generations
could lead to amplification of natural genetic variationand provide a source of heritable phenotypic variation(Vasileva et al., 1997; Johannes et al., 2009). Thus,
radiation effects may serve as a model for understanding
the mechanism producing phenotypic evolution of a
population in a stressed environment (Badyaev, 2005).
The effects of chronic radiation exposure on growth,development and reproduction have historically been thesubject of numerous studies (Marshall, 1962; Brown,
1964; Woodwell, 1967; Woodwell & Rebuck, 1967;
Geras’kin et al., 2007). These studies have shown adverse
effects on the fitness, longevity and health of livingorganisms (Zaire et al., 1997; Chen et al., 2001; Nicastro
et al., 2002; Møller et al., 2005; Ryabokon & Goncharova,
2006; Veremeyeva et al., 2010). Human populations
exposed to high radionuclide contamination have shown
increased rates of germline instability (Dubrova et al.,1998), transmissible, clastogenic factors from exposed
cells that damage unexposed cells (i.e. bystander effects)(Marozik et al., 2007) and elevated chromosome alterations (Neronova et al., 2003). Recently, Aghajanyan &Suskov (2009, 2010) demonstrated increased levels ofabnormal cells and chromosomes in children born after
the Chernobyl accident to irradiated fathers (i.e. liquidators) and non-irradiated mothers. These aberrations wereanalogous to the cytogenetic changes observed in children living in areas contaminated with radionuclides(Aghajanyan & Suskov, 2009, 2010). These results are
consistent with previous findings of children of Chernobyl liquidators conceived after the accident showing
high genomic DNA mutation levels that decreased withelapsed time between exposure and conception (Weinberg et al., 2001). These studies imply transgenerational
consequences of low-dose radiation exposure, although
the degree of the effect remains unclear (Slebos et al.,
2004; Furitsu et al., 2005).
Conditions in areas such as Chernobyl may serve as anatural laboratory for studying the effects of transgenerational genomic instability on fitness in populations
under chronic radiation exposure (Møller & Mousseau,
2006). On 26 April 1986, Reactor Four of the Chernobyl
nuclear plant exploded during a test of the reactor’sability to generate power during an unplanned shut
down. The resulting explosion released between
9.53 · 103 petabecquerel (PBq) and 1.25 · 104 PBq ofradionucleotides into the atmosphere (Konstantinov &Gonzalez, 1989; European Union, 1998; Chernobyl
Forum, 2005). Radioactive fallout dispersed across large
portions of the Soviet Union and Europe, particularly
Ukraine, Belarus and Russia (Shestopalov et al., 2003).
Most of the radionuclides dissipated within days, but

Caesium-137, Strontium-90 and Plutonium-239, among
others, still persist in the environment to this day(Yablokov et al., 2009).
Previous ecological studies in Chernobyl have demonstrated fitness and developmental consequences becauseof radiation exposure in a variety of vertebrate andinvertebrate species. Barn swallows (Hirundo rustica) in
the Chernobyl area have shown increased mutation rates(Ellegren et al., 1997; Møller et al., 2010), increased
frequency of partial albinism (Ellegren et al., 1997;
Møller & Mousseau, 2001), sperm abnormalities (Møller
et al., 2005, Møller et al., 2008a,b; Bonisoli-Alquati et al.,2011), increased DNA damage and oxidative stress(Bonisoli-Alquati et al., 2010a,b), reduced levels of circulating and stored antioxidants (Møller et al., 2005),
elevated levels of fluctuating asymmetry (FA) in taillength (Møller, 1993a,b) and smaller brains (Møller et al.,2011) in relation to radiation exposure. A study on stag
beetles (Lucanus cervus) in Chernobyl found that males inhighly contaminated sites had significantly elevatedlevels of FA in secondary sexual characters compared tomales from control sites, which affected mating status asa larger portion of asymmetric than symmetric males
were unmated (Møller, 2002). Additionally, studies ofhuman populations in areas such as the Semipalatinsk
Nuclear Test Site and the Techa River in Southern Urals,
Russia, where radioactive waste is dumped, showedsimilar increases in mutation rates in the germline as aresult of long-term radiation exposure (Dubrova et al.,
2002; Dubrova, 2006). Consistently, a decline in populations of both vertebrate and invertebrate species has
been shown in radioactively contaminated areas in
Chernobyl (Galván et al., 2011; Møller & Mousseau,
2007a,b, 2009, 2011a,b; Mousseau & Møller, 2011). Thisis particularly apparent in invertebrate species that live or
lay eggs in the topsoil where most of the radiation iscurrently located, suggesting a negative effect of direct
exposure to radiation (Møller & Mousseau, 2009).
The aim of this study was to assess the effect of parental
radiation exposure on offspring development undercommon garden conditions. We expected to see acomparable reduction in fitness across differing radiationlevels reflected in delayed development and reduced
body size at reproductive maturity in offspring derived
from high radiation sites. In addition, it could be
expected that disruption of development would be
buffered by longer developmental times (Shykoff &Møller, 1999). Therefore, the effect of parental exposure
to radioactive contamination is predicted to depend on
the developmental trajectory, as indexed by time tosexual maturity. We used variation in wing shape andsize and FA as proxies for the magnitude of developmental instability among offspring (Palmer & Strobeck,
1986, 2003; Klingenberg & McIntyre, 1998). FA reflects
the inability of the individual to undergo identicaldevelopment of bilateral traits under a wide range ofenvironmental and genetic stressors (Møller, 1993a,b;ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYParental radiation and offspring developmental instability

1151

Fig. 1 Locations of field collection sites andbackground radiation around Chernobyl.
Partly developed from Shestopalov (1996).
The collection site numbers are reported inTable 1.

Swaddle et al., 1994; Graham et al., 2010). Additionally,a negative correlation between degree of FA and fitness
components has been observed in many species (Møller
1992, Møller, 2002; Nosil & Reimchen, 2001; Mateos
et al., 2008; St-Amour et al., 2010), suggesting that FA
may be a reliable measure of phenotypic quality (Leary &Allendorf, 1989; Parsons, 1990; Møller & Thornhill,1998), although its ability to reliably reflect fitness has
been criticized in the literature (Palmer, 1999; - but see
Thornhill et al., 1999; Leung et al., 2003; Van Dongen,2006; Floate & Coghlin, 2010). We predict that theoffspring of parents from relatively high radiation sites
would show high levels of asymmetry in bilateral traits
compared to offspring from low radiation sites, as long asradiation did not cause increased rates of selective
mortality of asymmetric individuals, measured as thenumber of nymphs that failed to reach adulthood in theirradiated and nonirradiated groups (Floate & Fox, 2000).

coastal sand dunes, rivers and roadside verges. The
species has a univoltine life cycle with an obligate egg
diapause stage for overwintering. Nymphs typicallyemerge between May and June and develop through
4–5 nymphal instars. Adults occur from July to October.
We field collected C. albomarginatus from six different
locations in the Chernobyl area during 20–26 September
2009, across a range of radioactive contamination levels
by a factor of 1670 (Fig. 1; Table 1). The six locations
were
Chystohalivka(0.03 lSv h)1),
Rozizhdzhe)1(0.1 lSv h ), Vesniane (5.4 lSv h)1), Krasnytsia(10.72 lSv h)1), and two sites within the Red Forest(35.31 and 50.06 lSv h)1, respectively). Backgroundradiation levels were measured using a hand-held
dosimeter (Inspector; SE International, Summertown,
TN, USA). Although we did not measure the dose
received by individuals, we expect it to be highly
correlated with background radiation levels measured
at the collection sites owing to the short dispersal

Materials and methods
Study species and field procedures
Studies on the effects of irradiation on insect reproduction and development have shown Orthopterans to beparticularly radiosensitive (Willard & Cherry, 1975; Bakri
et al., 2005). The brown form of the lesser marsh
grasshopper (Chorthippus albomarginatus (Acrididae:
Orthoptera)) is a medium-sized grasshopper that has
undergone a northward range expansion within the last
10–15 years (Bellman, 1985; Vedenina & von Helversen,
2003). It is found in agricultural areas in dry and wet soil
throughout Europe from the southern half of Scandinavia and Finland to Spain and Italy (Bellman, 1985) along

Table 1 Number of families and average number of individuals
included in our study in each collection site. See Material andMethods for more details.

Site

Radiation(lSv h)1)

No. offamilies

Average number of
F1 progeny per family(minimum–maximum)

Chystohalivka (1)Rozizhdzhe (2)Vesniane (3)Krasnytsia (4)Red Forest 1(5)Red Forest 2 (6)

0.03
0.1
5.4
10.72
35.31
50.06

10
29
38
21
21
4

10.8 (0–16)
9.3 (0–17)
11.7 (5–20)
14 (4–26)
12.6 (5–27
16.5 (7–23)ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY1152

D. E. BEASLEY ET AL.

distance of our model species compared to the scale atwhich radioactive contamination levels vary (Fig. 1).
Individuals were collected from each collection site using
a standard insect sweep net. Females were placed
individually in Styrofoam cages (height ca. 12 cm, diameter ca. 8 cm) by collection site and provided withsterilized sand for ovipositing and fresh lettuce. Plastic
cups (height ca. 10 cm, diameter ca. 8 cm) were used to
enclose the top of the cages. Following removal frombreeding cages, the number of egg pods per female wascounted and they were placed in vials containing
moistened vermiculite to allow for the completion ofprediapause embryonic development.
Upon returning to the laboratory, we placed the egg
pods in a 4 C refrigerator to break diapause. Egg pods
were removed from the refrigerator following a 3-monthcold treatment and stored at room temperature (24 C) to
induce post-diapause embryonic development. Egg pods
were monitored twice daily, and newly emerged nymphs
were transferred to 9 cm · 9 cm · 8 cm plastic cages.
Cage density averaged nine nymphs per cage (range:
2–18 nymphs) over the duration of nymphal development. Families with large numbers of nymphs (> 10)
were divided into two cages. Cages were changed andfresh food provided twice weekly. Nymphs were fedorganic lettuce, carrots and wheat germ and provided
with a strip of unbleached paper towel for cover. We usedcarbon dioxide (CO2) to transfer nymphs to clean cageswith minimum mortality. The few individuals that diedbecause of accidental mishandling during the rearing
process were excluded from the analysis. Nymph cages
were transferred to an incubator set at a constanttemperature and photoperiod (28 C, 15:9 h LD) tocomplete development.
Life history estimates
We measured time to sexual maturity (time to maturity
hereafter) as the number of days from nymphal hatch to
final ecdysis. We also recorded body mass at sexual
maturity (body mass hereafter) and the pronotum length(accuracy 0.1 mm) as measures of adult body size.
Nymphs were monitored daily for final eclosure (maturity). Adults were removed from nymphal feeding cages
and weighed with a Sartorius Research electronic balance(Model Number: R160P) (0.001 g) within ca. 12 h after
emergence. We then placed adults in individual cageswith organic lettuce (3.0 g) and a carrot slice (9.0 g).
We transferred adults to clean cages and provided fresh
food twice weekly to maintain a clean environment.
Following death, adults were removed from cages andstored in 95% ethanol for subsequent measurements.
Wing measurements
The forewings and hind wings were removed from thethorax and wet-mounted on glass microscope slides with

distilled water and covered with a coverslip. Slides wereallowed to dry overnight at room temperature (24 C),
and then, coverslips were sealed to the slide with clear
nail polish.
Slides were scanned face down with an Epson V300
Photo Color Scanner set at a resolution of 2400 dpi.
Because measurement precision is important for the
analysis of FA (Palmer & Strobeck, 1986, 2003), on a
subsample of 20 individuals we scanned wings threetimes to assess scanning error using a Procrustes A N O V A
with wings as the main effect, number of scans as theerror effect and identity of specimens as the individual
effect (Klingenberg, 2011). All landmarks were located atwing–vein intersections and termination points (Fig. 2).
Most importantly, landmarks were anatomically homologous among all individuals, thus fulfilling the criterion
for Type I landmarks (Bookstein, 1991). Landmarks weredigitized on the left and right hind wing using TP S DI G 2 . 1 6
software (Rohlf, 2005). All wings within the sample were
measured three times to allow assessment of digitizing
error with a Procrustes A N O V A with wings as the side
effect, digitizing as the error effect and ID as theindividual effect (Klingenberg, 2011). Individuals weredigitized in random order, blind to information onparental exposure to radiation. The Procrustes A N O V A
found the between sides variation to far exceed themeasurement error because of the digitizing (F72 = 12.09,
P < 0.0001, r2 = 0.97) and scanning technique (F72 =17.20, P < 0.0001, r2 = 0.99) (Klingenberg, 2011). Additionally, we ran an analysis of error because of ourmounting technique by wet-mounting 54 wings on glass
microscope slides and covering them with a coverslip
before scanning. We repeated the process three times andthen measured the length of the radial vein (pixels) twice
for each wing using TP S DI G 2 . 1 6 software (Rohlf, 2005).
We used a one-way A N O V A with vein length as our
response variable and individual as our independent
variable to assess the repeatability of our measurements.
Measurement error because of mounting was very low
for both the left wing (F26 = 699.23, P < 0.0001, r2 =0.99) and right wing (F26 = 245.86, P < 0.0001,

Fig. 2 Hind wing of lesser marsh grasshopper (Chorthippus albomarginatus) showing landmark positions on wing veins (numbers)
and vein lengths (letters) selected for fluctuating asymmetry
measurements with image analysis.

ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYParental radiation and offspring developmental instability

r2 = 0.99) with repeatability in excess of 99.43%. In the
final analyses, we used mean landmark coordinates
calculated by averaging the three measurements per
landmark for each individual.
Statistical analysis
Only the individuals for whom both wings could bemeasured were included in the final analysis. Our
analysis included 454 individuals (223 males and 231
females). We extracted shape and size information fromthe raw data with the software MorphoJ (Klingenberg,2011) using a full Procrustes fit that controlled for wingorientation, position and size (Klingenberg & McIntyre,
1998; Klingenberg et al., 2002). We ran a Procrustes
A N O V A to obtain mean individual values of centroid size
as our measure of size variation and two measures ofshape: Procrustes distance and Mahalanobis distance.
Procrustes distance is the absolute measure of shape thattreats all aspects of shape variation equally (Klingenberg &Monteiro, 2005). Mahalanobis distance is a measure ofshape relative to the variation in the total sample, withemphasis on degree of invariance (Klingenberg &Monteiro, 2005). Thus, this measure can be interpreted
as the degree to which shape is unusual within the
population of individuals being analysed (Dryden &Mardia, 1998; Klingenberg & Monteiro, 2005; Breuker
et al., 2006). Centroid size is the square root of the sum ofsquared distances of a set of landmarks or the centre ofmass of the landmarks. It is the size measure used to scalea configuration of landmarks so they can be fitted in aframe of space, ultimately removing translation, size
differences and rotation from shape analysis (Rohlf, 1999;
Klingenberg & Monteiro, 2005). To further investigate
shape variation, we used the software tpsRelw (Rohlf,
2003) to calculate a matrix of shape variables, called
partial warp scores, which are coefficients indicating theposition of an individual relative to the consensus shape,and further reduced them by a relative warps analysis(Zelditch et al., 2004). Relative warps analysis is aprincipal component analysis of the partial warps anduniform components (Zelditch et al., 2004). The calculated relative warp scores (RWS) were subsequently usedas dependent variables in a M A N O V A (Soto et al., 2008).
The possibility of detecting a relationship between FA
and environmental stress is usually weak when testing
only one trait (Leung et al., 2000). Thus, we obtained
three measures of FA for our analysis. We defined wing
size FA as the absolute difference between the rightcentroid size value and the left centroid size value (|R–L|)(Takahashi et al., 2011). We also defined wing venation
FA as the absolute difference between the Euclidean
distances in Procrustes coordinates between landmarkson the right and left radial and one of the anal wing veins(Smith et al., 1997; Takahashi et al., 2011). We confirmedthat there was no significant directional asymmetry byperforming a t-test on the signed right minus left trait

1153

values (all P > 0.06) and confirmed the absence ofantisymmetry with tests of normality and visual inspection of data (all kurtosis > 1.00) (Palmer & Strobeck,
1986, 2003). We also confirmed no significant size
dependence of FA by regressing the unsigned, absolutedifference of the right and left trait values on trait size (allP > 0.46) (Palmer & Strobeck, 1986, 2003). The data
were inspected for statistically significant outliers, defined as being greater than three standard deviations
from the mean. Five individuals presenting significant
aberrant values for life history traits, shape or size were
excluded. We used the Kolmogorov–Smirnov test to
check the distribution of data. All measures of FA,
Procrustes distance and Mahalanobis distance were notnormally distributed, and we therefore log-transformed
them before analyses, although the log transformation
did not normalize the data.
We used a linear mixed model approach to test fordifferences in FA, wing shape and wing size according toradiation applying PROC MIXED in S A S 9.1 (SAS Institute, Cary, NC, USA). We separately tested three
response variables (Procrustes distance, Mahalanobis
distance and centroid size) one at a time as a functionof sex (as fixed factors), pronotum length, radiation level
and time to maturity (as covariates). In addition, we
included in the model the interaction between radiationand sex, radiation and time to maturity and sex and time
to maturity. We also included identity of the collection
site and identity of the family of origin (nested within thecollection site) as random effects to test the effect of bothshared genes and developmental environment on the
response variable. We used the Wald test (z) as the test ofsignificance for our random effects. This is equivalent to
the F test used to test significance of our fixed effects(SAS Institute Inc., 2008). We approximated degrees of
freedom using the Satterthwaite approximation.
We also used a linear mixed model approach to test
whether within-family mortality predicted wing shapevariation and FA within the family, while controlling for
nymphal density, as indexed by the total number ofindividuals per family. We tested average Procrustes
distance per family, average Mahalanobis distance perfamily and average measures of shape variation per family,
one at a time, as a function of radiation level and family
mortality. We also included in the model the interaction
between radiation level and mortality per family.
Additionally, we tested the independent effect ofradiation on time to maturity, family mortality, family
hatching success (measured as the number of individuals
that hatched over total number of eggs produced) andfecundity (measured as the total number of egg pods
produced) following the same linear mixed model
approach. Because nymphal density varied during therearing process, we also tested for independent effects ofdensity on shape variation and FA within the family. For
all linear mixed models, we applied an Akaike information criterion (AIC) approach for model selection,ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY1154

D. E. BEASLEY ET AL.

Fig. 3 Relationship between centroid size,
parental radiation exposure and time tomaturity. Results for both males and females
were combined because they showed a similar pattern. A surface was interpolated basedon the coefficients for the effects of radiation,
time to maturity and the interaction between
the two in the best-fit model. The coefficientsof the model are reported in Table 2.

sequentially removing the least significant terms, startingfrom the interactions. We removed a term from themodel only if removal did not increase the AIC values bymore than 2.00 (Burnham & Anderson, 1998).
For the M A N O V A , we applied PROC MIXED in S A S 9.1
and tested the relative warp scores as functions of thesame fixed factors, covariates and random effects appliedin the linear mixed model approach. We also tested theaverage relative warp scores per family as a function ofradiation level, family mortality estimated as the percentage of a family that died and the interaction between
radiation and mortality to determine whether family
mortality predicted shape variation within the family.

Results

males showed an increase in wing size with longer time
to maturity (coefficient = 5.34 · 10)3 (1.54 · 10)3 SE),t253 = 3.47, P < 0.01), whereas time to maturity did
not significantly affect wing size in females (coefficient =)1.85 · 10)3 (2.02 · 10)3 SE), t363 = )0.92, P = 0.36).
Overall, the wing size response to radiation depended on
the time at which the individual reached maturity(Fig. 3). Individuals that matured early had larger wings
with increasing radiation levels, whereas wing size in latematuring individuals decreased with increasing radiationlevels (Table 2).
In addition, neither collection site (z > 0.41, P > 0.26)
nor family (z > 1.26, P > 0.10) had a significant effect onwing size. Thus, the shared genes and environment were
not significantly associated with variation in wing size
among our experimental individuals.

Wing size
Radiation effects on wing size depended on whether theindividual was a male or female (Table 2), with wing
size increasing more with radiation levels in females(coefficient = 9.37 · 10)3 (3.51 · 10)3 SE), t291 = 2.67,
P < 0.01) compared to males (coefficient = 1.07 · 10)2
(3.65 · 10)3 SE), t299 = 2.94, P < 0.01). In addition,

Wing shape
We first analysed wing shape, as indexed by either
Procrustes distance (Table 3) or Mahalanobis distance(Table 4) as a function of radiation, sex, pronotum lengthand time to maturity. Radiation and time to maturity did
not have joint or independent effects on any measure of

Table 2 Results for linear mixed model for wing size variation as measured by centroid size. Statistically significant relationships (P < 0.05)
are marked in bold.
Final model
Pronotum
Radiation
Time to maturity
SexTime to maturity · radiation
Radiation · sex
Time to maturity · sex

CoefficientSE)3

7.11 · 109.37 · 10)3
)1.85 · 10)3)3

2.66 · 10
3.51 · 10)3
2.02 · 10)3

d.f.

F

P

1, 414
1, 291
1, 363
1, 404
1, 3191, 401
1, 404

7.14
7.95
1.37
11.43
7.44
4.41
12.92

< 0.001
0.01
0.24< 0.001
0.01
0.04< 0.001ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYParental radiation and offspring developmental instability

1155

Table 3 Linear mixed models for wing shape as measured by Procrustes distance. Dropped terms are reported in the order with which they
were removed from the model, with their associated statistics at removal.
Final model
Pronotum
Time to maturity
Radiation
Sex
Dropped terms
Radiation · sex
Time to maturity · sex
Time to maturity · radiation

CoefficientSE)4)3.50 · 10
)7.60 · 10)3
)1.40 · 10)3)2

1.37 · 10
5.09 · 10)3
2.14 · 10)3

d.f.

F

P

AIC

1, 411
1, 201
1, 9.1
1, 415

0.01
2.23
0.43
2.39

0.98
0.14
0.53
0.12

764.0

1, 411
1, 4101, 248

0.11
1.08
2.98

0.74
0.30
0.09

791.1
781.7
775.5

Table 4 Linear mixed models for wing shape as measured by Mahalanobis distance. Statistically significant relationships (P < 0.05) are marked
in bold. Dropped terms are reported in the order with which they were removed from the model, with their associated statistics at removal.
Final modelTime to maturity
Sex
Radiation
Pronotum
Dropped terms
Days to maturity · sex
Radiation · sex
Days to maturity · radiation

Coefficient)5.07 · 10SE)3

)8.90 · 10)4
)2.48 · 10)3

4.57 · 10)31.96 · 10)3
1.20 · 10)2

d.f.

F

P

AIC

1, 2481, 416
1, 10.6
1, 417

1.23
5.17
0.20
0.04

0.27
0.02
0.66
0.84

654.201, 407
1, 407
1, 305

0.48
0.59
2.17

0.49
0.44
0.14

683.00675.90666.60

Table 5 Results for multivariate mixed model for wing shape variation as measured by relative warp scores. Statistically significant
relationships (P < 0.05) are marked in bold. Dropped terms are reported in the order with which they were removed from the model, with theirassociated statistics at removal.
Final model
Pronotum
Radiation
Time to maturity
Sex
Dropped terms
Radiation · sex
Time to maturity · radiation
Time to maturity · sex

Coefficient)47.00 · 10
2.00 · 10)52.82 · 10)4SE
)44.02 · 10
5.70 · 10)55.70 · 10)5

wing shape (Tables 3 and 4). Males and females did not
differ significantly in wing shape, nor did they differ
significantly in how time to maturity was related towing shape (Tables 3 and 4). Time to maturity was
not significantly associated with shape of wings (Tables 3
and 4).
Males and females differed significantly in wing
shape, as indexed by RWS, but sex did not have aninteraction effect with radiation or time to maturity onwing shape. Radiation and time to maturity did nothave interaction or independent relationships with wing
shape (Table 5).
The collection site was not significantly related to wing
shape when measured as Procrustes distance (z > 0.00,
P > 0.22) or Mahalanobis distance (z > 0.07, P > 0.23).

d.f.

F

P

AIC

1, 3300
1, 3300
1, 3300
2, 3300

3.04
0.17
2.77
3.06

0.08
0.68
0.10
0.05

)10029.9

1, 3297
1, 3298
1, 3299

0.07
0.58
0.55

0.79
0.45
0.46

)10025.1)10027.0)10028.5

The relationship between collection site and wing shapevariation when measured as relative warp scores was
negligible. Additionally, the effect of family on shape was
not significant when measured as Procrustes distance(z > 0.10, P > 0.22), Mahalanobis distance (z > 0.86,
P > 0.19) or relative warp scores (z > 0.27, P > 0.39).
Radiation and family mortality were not jointly related
to average shape as indexed by average Procrustes distance (coefficient = 4.25 · 10)6 (1.20 · 10)5 SE), F97.5 =
0.13, P = 0.72), average Mahalanobis distance (coefficient = 6.30 · 10)5 (3.22 · 10)4 SE), F95.3 = 0.04, P =0.84) or average relative warp scores (coefficient =)3.05 · 10)6 (6.33 · 10)6 SE), F299 = 0.23, P = 0.63),
nor was radiation significantly independently related toany measures of shape variation (P > 0.55). Averageª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY1156

D. E. BEASLEY ET AL.

Table 6 Results for linear mixed model for wing shape fluctuating asymmetry as measured by distances between landmarks on the radial(LM1–LM2, LM2–LM3) and anal (LM5–LM6) veins. Akaike information criterion (AIC) model selection approach removes least significant
terms starting with interactions. Dropped terms are reported in the order with which they were removed from the model, with their associatedstatistics at removal.
Final model
Radial vein distance (LM 1–LM 2)Time to maturity
Sex
Radiation
Pronotum
Dropped terms
Radiation · sex
Days to maturity · radiation
Days to maturity · sex
Radial vein distance (LM 2–LM 3)
Time to maturity
Pronotum
Radiation
Dropped terms
Radiation · sex
Days to maturity · sex
Days to maturity · radiation
Sex
Anal vein distance (LM 5–LM 6)
Time to maturity
Radiation
Pronotum
Dropped terms
Days to maturity · sex
Radiation · sex
Days to maturity · radiation
Sex

CoefficientSE)8.08 · 10)3

1.45 · 10)2

)8.38 · 10)1)2.70 · 10)2

5.36 · 10)3
3.98 · 10)2

)1.09 · 10)2
)1.17 · 10)2
)7.43 · 10)3

1.28 · 10)3
1.64 · 10)3
3.96 · 10)2

1.36 · 10)2
3.08 · 10)2
4.63 · 10)3

9.15 · 10)3
3.11 · 10)3
2.06 · 10)2

shape variation decreased significantly with increasing
family mortality when measured as Mahalanobis distance (coefficient = )1.19 · 102 (4.79 · 10)3 SE), F97.7 =
6.18, P = 0.01), but not as Procrustes distance (coefficient = )2.80 · 10)4 (1.20 · 10)5 SE), F96.5 = 2.64, P =0.11) or relative warp scores (coefficient = 2.10 · 10)5(5.30 · 10)5 SE), F299 = 0.15, P = 0.70).
Wing fluctuating asymmetry
Radiation, sex and time to maturity were not related toany measure of wing FA (Table 6). Collection site(z > 0.14, P > 0.14) was not significantly related to any
measure of wing FA in addition to family not being
significantly related to FA.
Average wing FA, as indexed by distance between
landmarks on the radial vein, decreased significantlywith increasing family mortality (a: coefficient =)7.92 · 10)3 (3.72 · 10)2 SE), t97.9 = )2.13, P = 0.04;
b: coefficient = )1.30 · 10)2 (4.84 · 10)3 SE), t98 =)2.69, P = 0.01) and when indexed by distance between
landmarks on the anal vein (c: coefficient = )4.95 · 10)3(2.13 · 10)3 SE), t98 = )2.32, P = 0.02). Radiation was
not, independently or jointly, significantly related to any
measure of FA.

d.f.

F

P

AIC

1, 336
1, 413
1, 11.6
1, 414

0.31
0.95
2.44
0.46

0.58
0.33
0.14
0.50

1632.80

1, 411
1, 397
1, 413

0.00
0.05
0.22

0.95
0.82
0.64

1657.40
1649.90
1637.80

1, 207
1, 415
1, 46

0.64
0.14
2.57

0.42
0.12
0.70

1598.80

1, 4101, 4081, 214
1, 412

0.09
0.14
0.09
0.00

0.77
0.71
0.76
0.96

1625.00
1617.60
1612.50
1600.20

1, 2181, 50.1
1, 415

0.02
0.28
3.69

0.89
0.60
0.06

1266.60

1, 4081, 411
1, 226
1, 412

0.58
0.83
0.77
0.02

0.45
0.36
0.38
0.90

1294.101288.60
1281.201268.80

Life history
Radiation did not have an effect on time to maturity(coefficient = )9.57 · 10)3 (4.19 · 10)2 SE), t18.3 =)0.23, P = 0.82). Family mortality decreased significantlywith increasing radiation (coefficient = )4.45 · 10)1(1.64 · 10)1 SE), t117 = )2.71, P < 0.01), whereas family
hatching success decreased with increasing radiation but
not significantly (coefficient = )1.89 · 10)2 (1.02 · 10)2SE), t112 = )1.85, P = 0.07). Additionally, radiation did
not significantly affect fecundity (coefficient = 9.43 · 10)3
(9.82 · 10)3 SE), t5.63 = 0.96, P = 0.38).
Nymphal density did not have a significant effect on the
average wing size (F93.4 = 0.00, P = 0.96), average wing
shape as indexed by Procrustes distance (F99.9 =
1.15, P = 0.29), Mahalanobis distance (F97 = 2.73,
P = 0.10), relative warp scores (F700 = 0.02, P = 0.90) or
any measure of wing FA (P > 0.38).

Discussion
In this study of the transgenerational consequences ofenvironmental stress, we tested for effects of parental
radiation exposure on developmental instability of grasshopper offspring. We used three measures of wing shape,ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYParental radiation and offspring developmental instability

a measure of wing size and various measures of wing FA
to test whether the degree of developmental instability
was predicted by parental radiation exposure andwhether that effect depended on individual sex, bodysize and time to maturity. We also tested for effects ofparental radiation exposure on family mortality, family
hatching success and time to maturity in addition towhether mortality and nymphal density predicted developmental instability within families. If early developmental stages include more asymmetric individuals that
suffer from differential mortality, this may bias estimatesof the relationship between the environmental stressors,
such as radiation, and asymmetry (Møller, 1997).
Because we found weak, but nonsignificant effects ofradiation on fecundity and hatching failure, we can
exclude these factors as biasing our asymmetry estimates.
We also found that wing size decreased relative to bodysize with longer maturation time and increasing parental
radiation exposure. Thus, late maturing individuals bornto parents from high radiation sites were smaller thanlate maturing individuals whose parents came from low
radiation sites. Males responded to increasing parental
radiation exposure with increasing wing size, whereas
wing size decreased, but not significantly, in females.
Radiation exposure did not influence wing FA, independently or in combination with any other variable.
Previous studies on Orthopteran growth and development show that environmental variation significantly
affects the plasticity of adult size and time to eclosion(Moore, 1948; Willott & Hassall, 1998; Finch et al., 2008).
Additionally, this effect is greatly influenced by developmental stage. Individuals raised under optimal environmental conditions during early nymphal stages tend toreach adulthood with fewer moults and at a larger size
compared to individuals raised under suboptimal conditions regardless of whether conditions are reversed atlater nymphal stages (Flanagin et al., 2000; Miller et al.,
2001; Taniguchi & Tomioka, 2003). This suggests that thephysiological and hormonal events necessary for thetiming of eclosion and adult size are already fixed by thelater stages (Bradshaw & Johnson, 1995). Thus, if
radiation is interfering with these physiological events
early in development, it may be reflected in smaller adult
size and delayed eclosion. For example, the interaction
effect of radiation exposure and longer maturation time
resulting in smaller size at maturity may suggest that theoffspring’s physiological ability to metabolize and allocate
nutrients towards growth is developmentally unstable asa result of increasing parental radiation exposure.
In Orthopteran species, body size is significantly
correlated with fitness components. For example, Landa(1992) found that late hatching nymphs of Arphia
sulphurea and Chortophaga viridifasciata were smaller,
suffered higher overwinter mortality and reached reproductive maturity later compared to controls. Simmons(1995) found in the field cricket (Gryllus campestris) thatlarger males, as measured by their body length, were1157

more successful in obtaining mates as a result ofpossessing a larger harp which in turn affected the carrier
frequency of the male’s courtship song. In studies onbush crickets, larger males tend to produce more ejaculate compared to smaller males (Wedell, 1997). Additionally, larger size has been shown to contribute to anindividual’s resistance to parasitism (Danyk et al., 2000)
and is a determinant of fecundity in females (Fox &Czesak, 2000). Previous studies have demonstrated aphysiological trade-off between developmental rate andsize at maturity for various grasshopper species in
response to geographical and seasonal variation in foodquality (Asshoff & Hattenschwiler, 2005; Fielding &Defoliart, 2007; Jannot et al., 2009) that appears to occur
at the metabolic level (Behmer & Elias, 1999; Hahn,
2005). Møller & Mousseau (2011a,b) have suggested thatthe difference in susceptibility to radiation among invertebrates may in part be due to these differences inmetabolic rates relative to body size. Species withrelatively high metabolic rates may be more vulnerable
to radiation effects because of increased demand forantioxidant defence (Møller & Mousseau, 2011a,b). If
this is true also at the intra-specific level, faster development may increase susceptibility to oxidative stress andcompromise the metabolic capabilities and allocation of
resources for further development.
Additionally, poor maternal contribution to eggs as aconsequence of parental radiation exposure may be
exacerbating developmental selection pressure. During
oogenesis, females allocate resources including carbohydrates, proteins, lipids and water to eggs (Wheeler, 1996).
Grasshoppers obtain nutrients for oogenesis during adultfeeding and insufficient nourishment can adversely affectegg production and subsequent offspring development(Bellman, 1985; Wheeler, 1996; Fox & Czesak, 2000).
Egg size has been positively correlated with offspringsurvivorship and size in unfavourable environments(Braby, 1994; Mousseau & Fox, 1998). Mothers exposedto persistent oxidative stress may incur metabolic constraints that limit the acquisition of resources for eggs anddecrease the phenotypic quality of their offspring. Thus,
under conditions when mothers are limited in theavailability of important nutrients for eggs, as might be
the case in radioactively contaminated areas, offspring
are more vulnerable to environmental perturbations andmay need to compensate for reduced egg resources withlonger development time.
The negative trend between family mortality and FA
within families suggests that more asymmetric individuals suffered higher mortality rates compared to symmetrical individuals. Considering that offspring are often
produced in high numbers, one would expect a decreasein overall FA with increasing mortality as developmentally unstable individuals are removed by selection. Thisis especially so under chronic environmental stress whendevelopmental selection becomes more intense (Møller,
1997). Increased environmental stress may interfere withª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY1158

D. E. BEASLEY ET AL.

physiological mechanisms involved in maturation. For
example, studies on insect ecdysis (i.e. moult) found that
failure of ecdysis appears to occur because of disruptionof muscle contractions that are involved in working theindividual forward out of the old cuticle (Hughes, 1980;
Ewer et al., 1997; Park et al., 2003). Irradiation experiments found ecdysis failure to occur owing to abnormal
development of abdominal structures responsible for
expanding the abdomen and breaking the old cuticle,
similar to results found in insects treated with natural orsynthetic juvenile hormone analogues (Reynolds, 1980).
Under these conditions, adult eclosion failure was foundto increase with prolonged exposure to radiation (Beard,
1972; Sharma & Dwivedi, 1997; Faruki et al., 2007). In
one study of the tobacco hornworm Manduca sexta,
duration of development in offspring of irradiatedparents was roughly 10% longer and < 60% emerged
as adults compared to 90% of controls (Seth & Reynolds,
1993). If families are experiencing increased genomic
instability caused by parental radiation exposure, they
may experience greater selection pressure because of theincreased number of phenodeviants. If such deviance isreflected in physiological, immunological and behavioural function of the individual, insects may be particularly vulnerable to developmental selection pressure
from parental radiation exposure because malformed or
physiologically deviant individuals would be unable tosuccessfully eclose into adult form.
In conclusion, the effect of parental radiation exposure
can adversely affect offspring development and fitness
depending on developmental trajectories. However, caution in interpreting results is required. It is not certain
whether this effect was because of transgenerational
genomic instability or other influences such as maternal
contribution to eggs. We must also consider the possibility that the levels of radiation assessed in this study were
not sufficient for affecting FA in our model species. To
further assess the correlation of FA with fitness, futurestudies may wish to include more direct fitness parameters such as a female’s lifetime contribution to the next
generation in addition to life history traits such as escapebehaviour, mating success, immunocompetence andmaternal investment in egg content. Including more
replication in the experimental design so that therepeatability of such measures can be assessed will help
further our understanding of the relationship between
FA and fitness.

Acknowledgments
The authors would like to thank Rachel Co, Jason
Hubbard, Jen Fill and Heather Oswald for their assistancein mounting grasshopper wings and collecting morphometric data. In addition, we thank Igor Chizhevsky, Leonid
Milinevsky, Gennadi Milinevsky, the Chernobyl EcoCentre and the Chernobyl InterInform Agency for fieldassistance. We are sincerely grateful to Andrea Cardini forhis precious help with geometric morphometric analyses
and Stuart Reynolds for providing papers on insect adultecdysis. This study was partially funded through a GAANN
Fellowship to DEB and support from QIAGEN, GmbHthe Samuel Freeman Charitable Trust, CNRS and the
University of South Carolina to TAM and APM.

References
Aghajanyan, A. & Suskov, I. 2009. Transgenerational genomic
instability in children of irradiated parents as a result of theChernobyl nuclear accident. Mutat. Res.-Fundam. Mol. Mech.
Mutagen. 671: 52–57.
Aghajanyan, A.V. & Suskov, I.I. 2010. Genomic instability inchildren born after the Chernobyl nuclear accident (in vivo
and in vitro studies). Russ. J. Genet. 46: 740–749.
Asshoff, R. & Hattenschwiler, S. 2005. Growth and reproductionof the alpine grasshopper Miramella alpina feeding on CO2enriched dwarf shrubs at treeline. Oecologia 142: 191–201.
Badyaev, A.V. 2005. Stress-induced variation in evolution: frombehavioural plasticity to genetic assimilation. Proc. R. Soc. Lond.
Ser. B-Biol. Sci. 272: 877–886.
Bakri, A., Heather, N., Hendrichs, J. & Ferris, I. 2005. Fifty yearsof radiation biology in entomology: lessons learned fromIDIDAS. Ann. Entomol. Soc. Am. 98: 1–12.
Beard, R.L. 1972. Lethal action of UV irradiation on insects.
J. Econ. Entomol. 65: 650–654.
Behmer, S.T. & Elias, D.O. 1999. The nutritional significance ofsterol metabolic constraints in the generalist grasshopper
Schistocerca americana. J. Insect Physiol. 45: 339–348.
Bellman, H. 1985. A Field Guide to the Grasshoppers and Crickets ofBritain and Northern Europe. William Collins Sons & Co., London.
Bloem, S., Bloem, K.A., Carpenter, J.E. & Calkins, C.O. 1999.
Inherited sterility in codling moth (Lepidoptera : Tortricidae):effect of substerilizing doses of radiation on insect fecundity,
fertility, and control. Ann. Entomol. Soc. Am. 92: 222–229.
Bonisoli-Alquati, A., Voris, A., Mousseau, T.A., Møller, A.P.,
Saino, N. & Wyatt, M.D. 2010a. DNA damage in barnswallows (Hirundo rustica) from the Chernobyl region detectedby use of the comet assay. Comp. Biochem. Physiol. C-Toxicol.
Pharmacol. 151: 271–277.
Bonisoli-Alquati, A., Mousseau, T.A., Møller, A.P., Caprioli, M.
& Saino, N. 2010b. Increased oxidative stress in barn swallows
from the Chernobyl region. Comp. Biochem. Physiol. A-Mol.
Integr. Physiol. 155: 205–210.
Bonisoli-Alquati, A., Møller, A.P., Rudolfsen, G., Saino, N.,
Caprioli, M., Ostermiller, S. et al. 2011. The effects of radiation
on sperm swimming behavior depend on plasma oxidative
status in the barn swallow (Hirundo rustica). Comp. Biochem.
Physiol. A-Mol. Integr. Physiol. 159: 105–112.
Bookstein, F. 1991. Morphometric Tools for Landmark Data:
Geometry and Biology, 1st edn. Cambridge University Press,
Cambridge, MA.
Braby, M.F. 1994. The significance of egg size variation in
butterflies in relation to host plant quality. Oikos 71: 119–129.
Bradshaw, W.E. & Johnson, K. 1995. Initiation of metamorphosis in the pitcher-plant mosquito – effects of larval growth
history. Ecology 76: 2055–2065.
Breuker, C.J., Patterson, J.S. & Klingenberg, C.P. 2006. A single
basis for developmental buffering of drosophila wing shape.
PLoS ONE 1: (1) e7, 1–7.

ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYParental radiation and offspring developmental instability

Brown, S.O. 1964. Effects of continuous low intensity radiation
on successive generations of albino rat. Genetics 50: 1101–
1113.
Burnham, K.P. & Anderson, D.R. 1998. Model Selection andInference: A Practical Information-Theoretic Approach. SpringerVerlag, New York, NY.
Carpenter, J.E., Marti, O.G., Wee, S.L. & Suckling, D.M. 2009.
Cytological attributes of sperm bundles unique to f(1) progenyof irradiated male Lepidoptera: relevance to sterile insect
technique programs. Fla. Entomol. 92: 80–86.
Chen, W.L., Hwang, J.S., Hu, T.H., Chen, M.S. & Chang, W.S.P.
2001. Lenticular opacities in populations exposed to chronic
low-dose-rate gamma radiation from radiocontaminatedbuildings in Taiwan. Radiat. Res. 156: 71–77.
Chernobyl Forum, Chernobyl: the true scale of the accident.
20 years later a UN report provides definitive answers andways to repair lives, IAEA, WHO, UNDP, 2005.
Danyk, T., Johnson, D.L. & Mackauer, M. 2000. Parasitism of thegrasshopper Melanoplus sanguinipes by a sarcophagid fly,
Blaesoxipha atlanis: Influence of solitary and gregarious
development on host and parasitoid. Entomol. Exp. Appl. 94:
259–268.
Dryden, I. & Mardia, K.V. 1998. Statistical Shape Analysis. Wiley,
Chichester, NY.
Dubrova, Y.E. 2003. Radiation-induced transgenerational instability. Oncogene 22: 7087–7093.
Dubrova, Y.E. 2006. Genomic instability in the offspring of
irradiated parents: facts and interpretations. Russ. J. Genet. 42:
1116–1126.
Dubrova, Y.E., Plumb, M., Brown, J. & Jeffreys, A.J. 1998.
Radiation-induced germline instability at minisatellite loci. Int.
J. Radiat. Biol. 74: 689–696.
Dubrova, Y.E., Grant, G., Chumak, A.A., Stezhka, V.A. &Karakasian, A.N. 2002. Elevated minisatellite mutation ratein the post-Chernobyl families from Ukraine. Am. J. Hum.
Genet. 71: 801–809.
Eizirik, D.L. & Kisby, G.E. 1995. Cycad toxin-induced damage ofrodent and human pancreatic beta-cells. Biochem. Pharmacol.
50: 355–365.
Ellegren, H., Lindgren, G., Primmer, C.R. & Møller, A.P. 1997.
Fitness loss and germline mutations in barn swallows breedingin Chernobyl. Nature 389: 593–596.
European Union, 1998. Atlas of caesium deposition on Europe
after the Chernobyl accident. EU Publication EUR 16733,
Bruxelles, Belgium.
Ewer, J., Gammie, S.C. & Truman, J.W. 1997. Control of insect
ecdysis by a positive-feedback endocrine system: roles of
eclosion hormone and ecdysis triggering hormone. J. Exp. Biol.
200: 869–881.
Faruki, S.I., Das, D.R., Khan, A.R. & Khatun, M. 2007. Effects ofultraviolet (254 nm) irradiation on egg hatching and adultemergence of the flour beetles, Tribolium castaneum, T. confusum and the almond moth, Cadra cautella. J. Insect Sci. 7: 36.
Available online: insectscience.org ⁄ 7-36.
Fielding, D.J. & Defoliart, L.S. 2007. Growth, development, andnutritional physiology of grasshoppers from subarctic andtemperate regions. Physiol. Biochem. Zool. 80: 607–618.
Finch, O.D., Loffler, J. & Pape, R. 2008. Assessing the sensitivityof Melanoplus frigidus (Orthoptera : Acrididae) to different
weather conditions: a modeling approach focusing on development times. Insect Sci. 15: 167–178.

1159

Flanagin, V.L., Haase, S.P. & Juliano, S.A. 2000. Effects ofgrowth rates on development to metamorphosis in the lubber
grasshopper, Romalea microptera. Oecologia 125: 162–169.
Floate, K.D. & Coghlin, P. 2010. No support for fluctuating
asymmetry as a biomarker of chemical residues in livestock
dung. Can. Entomol. 142: 353–368.
Floate, K.D. & Fox, A.S. 2000. Flies under stress: a test offluctuating asymmetry as a biomonitor of environmental
quality. Ecol. Appl. 10: 1541–1550.
Fox, C.W. & Czesak, M.E. 2000. Evolutionary ecology
of progeny size in arthropods. Annu. Rev. Entomol. 45: 341–
369.
Furitsu, K., Ryo, H., Yeliseeva, K.G., Thuy, L.T.T., Kawabata, H.,
Krupnova, E.V. et al. 2005. Microsatellite mutations show noincreases in the children of the Chernobyl liquidators. Mutat.
Res., Genet. Toxicol. Environ. Mutagen. 581: 69–82.
Galván, I.G.I., Mousseau, T.A. & Møller, A.P. 2011. Bird
population declines due to radiation exposure at Chernobyl
are stronger in species with pheomelanin-based coloration.
Oecologia 165: 827–835.
Geras’kin, S.A., Evseeva, T.I., Belykh, E.S., Majstrenko, T.A.,
Michalik, B. & Taskaev, A.I. 2007. Effects on non-humanspecies inhabiting areas with enhanced level of naturalradioactivity in the north of Russia: a review. J. Environ.
Radioact. 94: 151–182.
Graham, J.H., Raz, S., Hel-Or, H. & Nevo, E. 2010. Fluctuating
asymmetry: methods, theory, and applications. Symmetry 2:
466–540.
Hahn, D.A. 2005. Larval nutrition affects lipid storage andgrowth, but not protein or carbohydrate storage in newly
eclosed adults of the grasshopper Schistocerca americana. J. Insect
Physiol. 51: 1210–1219.
Hoyes, K.P., Lord, B.I., McCann, C., Hendry, J.H. & Morris, I.D.
2001. Transgenerational effects of preconception paternal
contamination with Fe-55. Radiat. Res. 156: 488–494.
Hughes, T.D. 1980. Imaginal ecdysis of the desert locust,
Schistocerca gregaria. 1. Description of the behavior. Physiol.
Entomol. 5: 47–54.
Jaenisch, R. & Bird, A. 2003. Epigenetic regulation of gene
expression: how the genome integrates intrinsic and environmental signals. Nature Genet. 33: 245–254.
Jannot, J.E., Brinton, J., Kocot, K., Akman, O. & Juliano, S.A.
2009. Ontogenetic mechanisms underlying a geographic size
cline in a grasshopper, Romalea microptera. Ann. Entomol. Soc.
Am. 102: 467–475.
Johannes, F., Porcher, E., Teixeira, F.K., Saliba-Colombani, V.,
Simon, M., Agier, N. et al. 2009. Assessing the impact oftransgenerational epigenetic variation on complex traits. PLoS
Genet. 5: e10000530.
Klingenberg, C.P. 2011. Morphoj: an integrated software
package for geometric morphometrics. Mol. Ecol. Res. 11:
353–357.
Klingenberg, C.P. & McIntyre, G.S. 1998. Geometric morphometrics of developmental instability: analyzing patterns offluctuating asymmetry with Procrustes methods. Evolution 52:
1363–1375.
Klingenberg, C.P. & Monteiro, L.R. 2005. Distances and directions in multidimensional shape spaces: implications formorphometric applications. Syst. Biol. 54: 678–688.
Klingenberg, C.P., Barluenga, M. & Meyer, A. 2002. Shapeanalysis of symmetric structures: quantifying variationª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

1160

D. E. BEASLEY ET AL.

among individuals and asymmetry. Evolution 56: 1909–
1920.
Konstantinov, L.V. & Gonzalez, A.J. 1989. The radiologicalconsequences of the Chernobyl accident. Nuclear Safety 30:
53–69.
Koturbash, I., Rugo, R.E., Hendricks, C.A., Loree, J., Thibault,
B., Kutanzi, K. et al. 2006. Irradiation induces DNA damage
and modulates epigenetic effectors in distant bystander tissue
in vivo. Oncogene 25: 4267–4275.
Kovalchuk, O. & Baulch, J.E. 2008. Epigenetic changes andnontargeted radiation effects – is there a link? Environ. Mol.
Mutagen. 49: 16–25.
Landa, K. 1992. Seasonal declines in offspring fitness andselection for early reproduction in nymph-overwintering
grasshoppers. Evolution 46: 121–135.
Leary, R.F. & Allendorf, F.W. 1989. Fluctuating asymmetry as anindicator of stress – implications for conservation biology.
Trends Ecol. Evol. 4: 214.
Leung, B., Forbes, M.R. & Houle, D. 2000. Fluctuating asymmetry as a bioindicator of stress: comparing efficacy ofanalyses involving multiple traits. Am. Nat. 155: 101–115.
Leung, B., Knopper, L.D. & Mineau, P. 2003. A criticalassessment of the utility of fluctuating asymmetry as abioindicator of anthropogenic stress. In: Developmental Instability:
Causes and Consequences (M. Polak, ed.), pp. 415–426. Oxford
University Press, Oxford, UK.
Luke, G.A., Riches, A.C. & Bryant, P.E. 1997. Genomic instability
in haematopoietic cells of f-1 generation mice of irradiated maleparents. Mutagenesis 12: 147–152.
Lyon, M.F. & Renshaw, R. 1988. Induction of congenitalmalformation in mice by parental irradiation – transmission tolater generations. Mutat. Res. 198: 277–283.
Marozik, P., Mothersill, C., Seymour, C.B., Mosse, I. & Melnov,
S. 2007. Bystander effects induced by serum from survivors of
the Chernobyl accident. Exp. Hematol. 35: 55–63.
Marshall, J.S. 1962. Effects of continuous gamma-radiation on
intrinsic rate of natural increase of Daphnia pulex. Ecology 43:
598–607.
Mateos, C., Alarcos, S., Carranza, J., Sanchez-Prieto, C.B. &Valencia, J. 2008. Fluctuating asymmetry of red deer antlers
negatively relates to individual condition and proximity toprime age. Anim. Behav. 75: 1629–1640.
Miller, W.A., Hatle, J.D. & Borst, D.W. 2001. The second half ofthe molt cycle in lubber grasshoppers is developmentally
inflexible. Am. Zool. 41: 1527.
Mishra, P.K., Bhargava, A., Raghuram, G.V., Gupta, S., Tiwari,
S., Upadhyaya, R. et al. 2009. Inflammatory response toisocyanates and onset of genomic instability in cultured
human lung fibroblasts. Genet. Mol. Res. 8: 129–143.
Møller, A.P. 1992. Patterns of fluctuating asymmetry in weapons evidence for reliable signaling of quality in beetle horns and bird
spurs. Proc. R. Soc. Lond. Ser. B-Biol. Sci. 248: 199–206.
Møller, A.P. 1993a. Fluctuating asymmetry. Nature 363: 217–217.
Møller, A.P. 1993b. Morphology and sexual selection in the barnswallow Hirundo rustica in Chernobyl, Ukraine. Proc. R. Soc.
Lond. B-Biol. Sci. 252: 51–57.
Møller, A.P. 1997. Developmental selection against developmentally unstable offspring and sexual selection. J. Theor. Biol.
185: 415–422.
Møller, A.P. 2002. Developmental instability and sexual selection in stag beetles from Chernobyl and a control area.
Ethology 108: 193–204.

Møller, A.P. & Mousseau, T.A. 2001. Albinism and phenotype ofbarn swallows (Hirundo rustica) from Chernobyl. Evolution 55:
2097–2104.
Møller, A.P. & Mousseau, T.A. 2006. Biological consequences ofChernobyl: 20 years on. Trends Ecol. Evol. 21: 200–207.
Møller, A.P. & Mousseau, T.A. 2007. Species richness andabundance of forest birds in relation to radiation at Chernobyl.
Biol. Lett. 3: 483–486.
Møller, A.P. & Mousseau, T.A. 2009. Reduced abundance ofinsects and spiders linked to radiation at Chernobyl 20 years
after the accident. Biol. Lett. 5: 356–359.
Møller, A.P. & Mousseau, T.A. 2011a. Conservation consequences of Chernobyl and other nuclear accidents. Biol. Cons.
144: 2787–2798.
Møller, A.P. & Mousseau, T.A. 2011b. Efficiency of bio-indicators for low-level radiation under field conditions. Ecol. Indic.
11: 424–430.
Møller, A.P. & Thornhill, R. 1998. Bilateral symmetry and sexual
selection: a meta-analysis. Am. Nat. 151: 174–192.
Møller, A.P., Mousseau, T.A., Milinevsky, G., Peklo, A.,
Pysanets, E. & Szep, T. 2005. Condition, reproduction andsurvival of barn swallows from Chernobyl. J. Anim. Ecol. 74:
1102–1111.
Møller, A.P., Karadas, F. & Mousseau, T.A. 2008a. Antioxidants
in eggs of great tits Parus major from Chernobyl and hatching
success. J. Comp. Physiol. B-Biochem. Syst. Environ. Physiol. 178:
735–743.
Møller, A.P., Mousseau, T.A., Lynnn, C., Ostermiller, S. &Rudolfsen, G. 2008b. Impaired swimming behaviour andmorphology of sperm from barn swallows Hirundo rustica in
Chernobyl. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 650:
210–216.
Møller, A.P., Erritzoe, J., Karadas, F. & Mousseau, T.A. 2010.
Historical mutation rates predict susceptibility to radiation in
Chernobyl birds. J. Evol. Biol. 23: 2132–2142.
Møller, A.P., Bonisoli-Alquati, A., Rudolfsen, G. & Mousseau,
T.A. 2011. Chernobyl birds have smaller brains. PLoS ONE 6:
e16862.
Monaghan, P., Metcalfe, N.B. & Torres, R. 2009. Oxidative stressas a mediator of life history trade-offs: mechanisms, measurements and interpretation. Ecol. Lett. 12: 75–92.
Moore, H.W. 1948. Variations in fall embryological developmentin three grasshopper species. Can. Entomol. 80: 83–88.
Morgan, W.F. 1999. The use of chromosome rearrangements toevaluate genomic instability. Radiat. Res. 151: 111–112.
Mousseau, T.A. & Fox, C.W. 1998. The adaptive significance ofmaternal effects. Trends Ecol. Evol. 13: 403–407.
Mousseau, T.A. & Møller, A.P. 2011. Landscape portrait: a lookat the impacts of radioactive contaminants on Chernobyl’s
wildlife. Bull. Atom. Scient. 67: 38–46.
Natarajan, A.T. 2006. Induced transgenerational genetic effects
in rodents and humans. J. Radiat. Res. 47: B39–B43.
Neronova, E., Slozina, N. & Nikiforov, A. 2003. Chromosome
alterations in cleanup workers sampled years after Chernobyl
accident. Radiat. Res. 160: 46–51.
Nicastro, A.J., Vreeland, R.H. & Rosenzweig, W.D. 2002. Limits
imposed by ionizing radiation on the long-term survival oftrapped bacterial spores: beta radiation. Int. J. Radiat. Biol. 78:
891–901.
Nomura, T. 1982. Parental exposure to x-rays and chemicals
induces heritable tumors and anomalies in mice. Nature 296:
575–577.

ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYParental radiation and offspring developmental instability

North, D.T. & Holt, G. 1968. Inherited sterility in progeny ofirradiated male cabbage loopers. J. Econ. Entomol. 61: 928–931.
North, D.T. & Holt, G.G. 1969. Population suppression bytransmission of inherited sterility to progeny of irradiatedcabbage loopers Trichoplusia ni. Can. Entomol. 101: 513–520.
Nosil, P. & Reimchen, T.E. 2001. Tarsal asymmetry, nutritional
condition, and survival in water boatmen (Callicorixa vulnerata). Evolution 55: 712–720.
Palmer, A.R. 1999. Detecting publication bias in meta-analyses:
a case study of fluctuating asymmetry and sexual selection.
Am. Nat. 154: 220–233.
Palmer, A.R. & Strobeck, C. 1986. Fluctuating asymmetry –measurement, analysis, patterns. Annu. Rev. Ecol. Syst. 17: 391–
421.
Palmer, A.R. & Strobeck, C. 2003. Fluctuating asymmetry
analysis revisited. In: Developmental Instability: Causes andConsequences (M. Polak, ed.), pp. 279–319. Oxford University
Press, Oxford, UK.
Park, J.H., Schroeder, A.J., Helfrich-Forster, C., Jackson, F.R. &Ewer, J. 2003. Targeted ablation of ccap neuropeptidecontaining neurons of Drosophila causes specific defects inexecution and circadian timing of ecdysis behavior. Development 130: 2645–2656.
Parsons, P.A. 1990. Fluctuating asymmetry – an epigenetic
measure of stress. Biol. Rev. Cambridge Philos. Soc. 65: 131–145.
Reynolds, S.E. 1980. Integration of behaviour and physiology inecdysis. Adv. Insect Physiol. 15: 475–595.
Riley, P.A. 1994. Free-radicals in biology – oxidative stress andthe effects of ionizing-radiation. Int. J. Radiat. Biol. 65: 27–33.
Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. & Bonner,
W.M. 1998. DNA double-stranded breaks induce histone h2ax
phosphorylation on serine 139. J. Biol. Chem. 273: 5858–5868.
Rohlf, F.J. 1999. Shape statistics: procrustes superimpositions
and tangent spaces. J. Classif. 16: 197–223.
Rohlf, F.J. 2003. Tpsrelw, relative warps analysis, version 1.36.
In: Department of Ecology and Evolution, State University of
New York at Stony Brook.
Rohlf, F.J. 2005. Tpsdig, digitize landmarks and outlines, version
2.05. In: Department of Ecology and Evolution, State University of New York at Stony Brook.
Rugo, R.E., Secretan, M.B. & Schiestl, R.H. 2002. X radiation
causes a persistent induction of reactive oxygen species and adelayed reinduction of tp53 in normal human diploid fibroblasts. Radiat. Res. 158: 210–219.
Ryabokon, N.I. & Goncharova, R.I. 2006. Transgenerational
accumulation of radiation damage in small mammals chronically exposed to Chernobyl fallout. Radiat. Environ. Biophys.
45: 167–177.
SAS Institute Inc. 2008. S A S ⁄ S T A T 9.2 User’s Guide. SAS InstituteInc., Cary, NC.
Seth, R.K. & Reynolds, S.E. 1993. Induction of inherited sterility
in the tobacco hornworm Manduca sexta (Lepidoptera, Sphingidae) by substerilizing doses of ionizing-radiation. Bull.
Entomol. Res. 83: 227–235.
Sharma, M.K. & Dwivedi, S.C. 1997. Investigation on the effects
of ultraviolet and infra-red light on the life cycle of Callosobruchus chinensis linn. J. Adv. Zool. 18: 27–31.
Shestopalov, V.M. 1996. Atlas of Chernobyl Exclusion Zone.
Ukrainian Academy of Science, Kiev.
Shestopalov, V.M., Kashparov, V.A. & Ivanov, Y.A. 2003.
Radionuclide migration into the geological environment and1161

biota after the Chernobyl accident. Environ. Sci. Pollut. Res. 3:
9–47.
Shykoff, J.A. & Møller, A.P. 1999. Fitness and asymmetry under
different environmental conditions in the barn swallow. Oikos
86: 152–158.
Simmons, L.W. 1995. Correlates of male quality in the field
cricket, Gryllus campestris l: age, size, and symmetry determine
pairing success in field populations. Behav. Ecol. 6: 376–381.
Sinclair, W.K. 1964. X-ray-induced heritable damage (smallcolony formation) in cultured mammalian cells. Radiat. Res.
21: 584–611.
Slebos, R.J.C., Little, R.E., Umbach, D.M., Antipkin, Y.,
Zadaorozhnaja, T.D., Mendel, N.A. et al. 2004. Mini-and
microsatellite mutations in children from Chernobyl accident
cleanup workers. Mutat. Res., Genet. Toxicol. Environ. Mutagen.
559: 143–151.
Smith, D.R., Crespi, B.J. & Bookstein, F.L. 1997. Fluctuating
asymmetry in the honey bee, Apis mellifera: effects of ploidy
and hybridization. J. Evol. Biol. 10: 551–574.
Soto, I.M., Carreira, V.P., Soto, E.M. & Hasson, E. 2008. Wing
morphology and fluctuating asymmetry depend on the hostplant in cactophilic Drosophila. J. Evol. Biol. 21: 598–609.
St-Amour, V., Garner, T.W.J., Schulte-Hostedde, A.I. & Lesbarreres, D. 2010. Effects of two amphibian pathogens on thedevelopmental stability of green frogs. Conserv. Biol. 24: 788–
794.
Swaddle, J.P., Witter, M.S. & Cuthill, I.C. 1994. The analysis offluctuating asymmetry. Anim. Behav. 48: 986–989.
Takahashi, K.H., Daborn, P.J., Hoffmann, A.A. & TakanoShimizu, T. 2011. Environmental stress-dependent effects ofdeletions encompassing hsp70ba on canalization and quantitative trait asymmetry in Drosophila melanogaster. PLoS ONE 6:
e17295.
Taniguchi, N. & Tomioka, K. 2003. Duration of development andnumber of nymphal instars are differentially regulated byphotoperiod in the cricket Modicogryllus siamensis (Orthoptera :Gryllidae). Eur. J. Entomol. 100: 275–281.
Thornhill, R., Møller, A.P. & Gangestad, S.W. 1999. The
biological significance of fluctuating asymmetry and sexual
selection: a reply to Palmer. Am. Nat. 154: 234–241.
Van Dongen, S. 2006. Fluctuating asymmetry and developmental instability in evolutionary biology: past, present and future.
J. Evol. Biol. 19: 1727–1743.
Vasileva, L.A., Ratner, V.A. & Bubenshchikova, E.V. 1997.
Stress induction of retrotransposon transpositions in Drosophila: reality of the phenomenon, characteristic features,and possible role in rapid evolution. Genetika 33: 1083–
1093.
Vedenina, V.Y. & von Helversen, O. 2003. Complex courtship ina bimodal grasshopper hybrid zone. Behav. Ecol. Sociobiol. 54:
44–54.
Veremeyeva, G., Akushevich, I., Pochukhailova, T., Blinova, E.,
Varfolomeyeva, T., Ploshchanskaya, O. et al. 2010. Long-term
cellular effects in humans chronically exposed to ionizing
radiation. Health Phys. 99: 337–346.
Wedell, N. 1997. Ejaculate size in bushcrickets: The importanceof being large. J. Evol. Biol. 10: 315–325.
Weinberg, H.S., Korol, A.B., Kirzhner, V.M., Avivi, A., Fahima,T., Nevo, E. et al. 2001. Very high mutation rate in offspring of
Chernobyl accident liquidators. Proc. R. Soc. Lond. Ser. B-Biol.
Sci. 268: 1001–1005.

ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY1162

D. E. BEASLEY ET AL.

Wheeler, D. 1996. The role of nourishment in oogenesis. Annu.
Rev. Entomol. 41: 407–431.
Willard, W.K. & Cherry, D.S. 1975. Comparative radiosensitivity
in class Insecta. J. Theor. Biol. 52: 149–158.
Willott, S.J. & Hassall, M. 1998. Life-history responses of British
grasshoppers (Orthoptera: Acrididae) to temperature change.
Funct. Ecol. 12: 232–241.
Wong, K.K., Maser, R.S., Bachoo, R.M., Menon, J., Carrasco,
D.R., Gu, Y.S. et al. 2003. Telomere dysfunction and atm
deficiency compromises organ homeostasis and accelerates
ageing. Nature 421: 643–648.
Woodwell, G.M. 1967. Radiation and patterns of nature. Science
156: 461.
Woodwell, G.M. & Rebuck, A.L. 1967. Effects of chronic gamma
radiation on structure and diversity of an oak-pine forest. Ecol.
Monogr. 37: 53.
Wu, C.C., Liu, M.T., Chang, Y.T., Fang, C.Y., Chou, S.P., Liao,
H.W. et al. 2010. Epstein-barr virus dnase (bglf5) induces

genomic instability in human epithelial cells. Nucleic Acids Res.
38: 1932–1949.
Yablokov, A.V., Nesterenko, V.B. & Nesterenko, A.V. 2009.
Atmospheric, water, and soil contamination after Chernobyl. In: Chernobyl: Consequences of the Catastrophe for People
and the Environment (J.D. Sherman-Nevinger I, ed.), pp.
223–236. New York Academy of Sciences, Boston, MA,
USA.
Zaire, R., Notter, M., Riedel, W. & Thiel, E. 1997. Unexpected
rates of chromosomal instabilities and alterations of hormone levels in Namibian uranium miners. Radiat. Res. 147:
579–584.
Zelditch, M.L., Swiderski, D.L., Sheets, H.D. & Fink, W.L. 2004.
Geometric Morphometrics for Biologists: A Primer. Elsevier
Academic Press, New York and London.
Received 14 September 2011; revised 2 March 2012; accepted 5 March2012ª 2012 THE AUTHORS. J. EVOL. BIOL. 25 (2012) 1149–1162
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2012 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY