Behavioral Ecology
doi:10.1093/beheco/arh138
Advance Access publication 5 August 2004
Larval food source promotes cyclic seasonal
variation in polyandry in the moth
Lobesia botrana
Luis M. Torres-Vila,a M. Carmen Rodrı́guez-Molina,b Miguel McMinn,c and Ana Rodrı́guez-Molinac
Servicio de Sanidad Vegetal, Consejerı́a de Agricultura y Medio Ambiente, Avda. de Portugal s/n,
E-06800 Mérida, Badajoz, Spain, bServicio de Investigación y Desarrollo Tecnológico (SIA),
Consejerı́a de Agricultura y Medio Ambiente, Mérida, Badajoz, Spain, and cSkua Gabinete de
Estudios Ambientales SL, Palma de Mallorca, Illes Balears, Spain
a
In Mediterranean vineyards the European grapevine moth, Lobesia botrana, usually completes three non-overlapping larval
generations per year. Larvae feed on inflorescences and unripe or ripe grapes. Previous work has shown that the larval food
source has a huge effect on adult size and fitness. We investigated if larval food also affects the propensity of females to mate
polyandrously. Levels of polyandry were monitored with baited traps in a vineyard for two years and then compared with data
from laboratory trials using vine-reared individuals. In the field, polyandry levels followed a cyclic annual pattern associated with
the larval food source. Large females that fed on ripe grapes exhibited significantly higher levels of polyandry than smaller
females that fed on inflorescences. Females that fed on unripe grapes showed intermediate levels of remating. Polyandry also
tended to increase later in each flight. Laboratory trials confirmed a direct effect of larval feeding on levels of polyandry. Overall,
these results showed that the seasonal variation in polyandry exhibited by L. botrana in the field occurs, at least in part, because of
food-derived changes regulating female size. Results also suggested that larval feeding could shape sexual size dimorphism and
polyandry levels. Larval nutrition should therefore be considered as an important ecological factor by those studying the
evolutionary significance of polyandry and sperm competition in the Lepidoptera. Key words: body size, female age, larval feeding,
Lobesia botrana, polyandry, sexual size dimorphism. [Behav Ecol]
ultiple mating of females with several males (polyandry)
is a crucial aspect of insect mating systems that has
generated important debate in the last decade because of its
fitness implications. Polyandry is a prerequisite for the
evolution of sperm competition and cryptic female choice
(Birkhead and Møller, 1998; Choe and Crespi, 1997;
Drummond, 1984; Eberhard, 1996; Simmons, 2001; Thornhill
and Alcock, 1983). Four key benefits are often invoked to
explain the widespread occurrence of polyandry across most
insect taxa. First, females obtain male-transferred nutrients at
mating that enhance their fecundity, offspring fitness, or both
(Arnqvist and Nilsson, 2000). Second, polyandry ensures that
females store sufficient sperm to fertilize all their eggs (Ridley,
1988). Third, it allows for cryptic female choice and
preferential use of either genetically more compatible sperm
or the sperm of a fitter male to increase mean offspring fitness
( Jennions and Petrie, 2000; Zeh and Zeh, 2001, 2003).
Fourth, multiple paternity elevates the genetic variability of
progeny, increasing the chances some offspring will cope with
unpredictable or fluctuating environments (Yasui, 1998).
Several potential costs to female remating may, however,
counteract these polyandry-derived benefits. These include
exposure to male-borne parasites and diseases, loss of time or
energy, and an increased risk of physical injury and predation.
Polyandry levels in insects are known to be influenced by
both genetic and environmental factors. First, a heritable
genetic component to polyandry, an indispensable prerequisite for the selection and evolution of this trait, has been
M
Address correspondence to L. M. Torres-Vila. E-mail: luis.torres@
aym.juntaex.es
Received 21 January 2004; revised 14 April 2004; accepted 31 May
2004.
reported (Simmons, 2003; Torres-Vila et al., 2001, 2002;
Wedell et al., 2002;). Second, polyandry in lepidopterans has
shown to be affected by population age structure, voltinism,
weather variables, population density, operational sex ratios,
host plant maturity, and food quality (Drummond, 1984). Of
these environmental factors, larval feeding is considered to be
especially important in holometabolous insects because it
strongly affects adult size. Adult body size might affect
polyandry in two ways. First, several studies have demonstrated
that larger males deliver larger spermatophores that reduce
post-mating receptivity and female propensity to remate
(Drummond, 1984; Torres-Vila et al., 1995 and references
therein, but see also Marshall and McNeil, 1989). Second,
there is also evidence that larger females show a greater
propensity to remate (Bergström et al., 2002; Miyahara, 1978;
Torres-Vila et al., 1997a).
The European grapevine moth, Lobesia botrana Den. and
Schiff. (Lep: Tortricidae), is an ideal species to study how larval
nutrition might influence polyandry. It is a predominantly
monandrous species in which a reduced frequency of polyandry
has proved to be modulated by both genetic (Torres-Vila et al.,
2002) and environmental or physiological factors (Torres-Vila
et al., 1997a). Recent research has shown that when L. botrana
develops on vine, its typical host plant, adults exhibit a huge
seasonal variation in body size. This variation is related to the
quality of the food available for larvae, which depends on vine
phenology (Torres-Vila et al., 1999). Adult size has strong
consequences for fitness. Large size enhances lifetime fecundity and egg size in females, lifetime spermatophore number
and size in males, and longevity in both sexes (Torres-Vila et al.,
1999; Torres-Vila and Rodrı́guez-Molina, 2002).
The seasonal variation in L. botrana adult size led Torres-Vila
et al. (1999) to hypothesise that larval feeding experience
Behavioral Ecology vol. 16 no. 1 International Society for Behavioral Ecology 2005; all rights reserved.
Torres-Vila et al.
•
Polyandry variation in Lobesia botrana
might also influence polyandry, as in this species both female
size and male size are closely related to female remating
behavior (Torres-Vila et al., 1997a). These authors pointed
out that the actual effect of seasonal variation in adult size on
polyandry is, however, not immediately obvious, as female size
and male size have conflicting effects on female remating. Any
increase in female reproductive tract size could be fully
balanced by an increase in the size of spermatophores
delivered by males, so remating frequency could remain
unchanged from one generation to another.
In this study we investigated whether the phenological
development of the vine throughout the growing season has
a significant effect on seasonal polyandry levels in L. botrana.
Our prediction was the occurrence of a positive association
between polyandry and larval feeding via its effect on
increased female size. Additionally, we investigated whether
patterns of variation in polyandry in field conditions could be
reproduced in a laboratory strain where we manipulated larval
food supply but kept the environmental factors constant for
the resultant adults. We simulated the three typical larval
generations of the moth on vine by rearing larvae on
inflorescences and on unripe and ripe grapes, to further
assess in a controlled environment the subsequent levels of
polyandry.
METHODS
Study species
Lobesia botrana is a major vine pest in the Palaearctic region. In
southern temperate areas, this moth usually completes three
annual non-overlapping generations on vine, although
a partial fourth generation is not exceptional. Larvae typically
develop on inflorescences, unripe grapes, and ripening-ripe
grapes, giving rise to the second, third, and first annual
flights, respectively. Adult emergence is protandrous. The
larval generation and adult flight order is as indicated because
offspring from third-flight individuals overwinter as diapausing pupae and emerge the following spring (Bovey, 1966;
Coscollá, 1997; Torres-Vila, 2000).
Field population
The mating dynamics of L. botrana were investigated in the
field in a 0.05-ha vine experimental plot placed within a 2.5-ha
vineyard (cultivar Macabeo) at the Finca la Orden, Badajoz,
southwestern Spain. Insecticide treatments were not applied
in the experimental plot during the study. Adults were
trapped from March to October in two consecutive years
(2000 and 2001) and collected twice weekly with two 2-l baited
plastic traps placed 50 m apart. The bait used was slightly
modified from Coscollá (1997) and consisted of red wine,
vinegar, commercial sucrose (600 cc þ 150 cc þ 400 g), and
water to complete 5 l of solution. Traps were initially baited
with 1-l of solution and refilled when necessary, to compensate
for evaporation and degradation losses. All females collected
in each sampling date (or a random subsample if the number
caught was too high) were carefully dissected under a stereomicroscope, and we counted the number of spermatophores
in the bursa copulatrix. We also recorded female reproductive
age according to the Feldhedge and Louis scale (Coscollá,
1997). Females were scored as virgin (1) or mated with either
totally full ovaries (2), half-depleted ovaries (3), or empty
ovaries (4). Intermediate reproductive-age classes were used
when necessary. As an estimate of adult body size we measured
the mid-femur length (mm) with a micrometer to the nearest
0.01 mm. Two pheromone delta-traps were also placed in the
experimental plot to monitor male population dynamics.
115
Laboratory strain
Preliminary field results suggested that the development of
vine reproductive organs, because it modified the diet
available for larvae, was involved in the observed seasonal
changes in the occurrence of polyandry. We therefore
conducted a laboratory experiment to directly confirm the
effect of larval feeding on polyandry, while controlling the
environmental conditions in which adults reproduced. We
obtained insects from a laboratory strain reared on an
artificial diet (Stockel et al., 1989). To produce adults developed on vine, three artificial infestations with neonate larvae
were conducted simulating the first (on inflorescences),
second (on unripe grapes), and third (on ripening-ripe
grapes) larval generations. The vine plants used were at
phenological stages 17, 32, and 35–36, respectively (Eichhorn
and Lorenz, 1977). Infestations on inflorescences and unripe grapes were conducted in the field, while that on ripe
grapes was performed on excised grape clusters in the laboratory under long day photoperiod (16:8 L:D) to avoid
pupal diapause. Additional information is given in TorresVila (1996) and Torres-Vila et al. (1997b, 1999). To ensure
virginity of moths, pupae were isolated in glass tubes
stoppered with cardboard plugs. Newly emerged moths (,
24-h old) were collected daily, sexed, and weighed to the
nearest 0.1 mg.
We created pairs consisting of a two-day-old virgin female
and one-day-old virgin male, both individuals reared in larval
stage on the same diet. Pairs were caged in 22-ml clear plastic
containers that acted as both mating and oviposition
chambers, and they were provided with water ad libitum.
Adults were tested in a controlled environment room at 22 6
1 C, 60 6 20% relative humidity and an L(15 þ 1):D8
photoperiod. The first 15 photophase hours (day) were at
a 1000 lux luminosity and the last hour (simulating dusk) was
at 25 lux luminosity. Artificial dusk was used to observe adult
behavior because this is when vital activities (flight, calling,
mating, egg-laying, or feeding) take place in L. botrana (Bovey,
1966). The sexual activity of moths was continuously observed
during the first dusk period and mated pairs were noted. The
following morning, unmated pairs (, 5%) were rejected and
mated females were isolated to be observed at dusk
throughout their lifetime. Females that resumed calling
(recalled) in a given dusk period were provided with another
virgin one-day-old male. If the female did not remate, the
male was removed after dusk to avoid possible unobserved
matings, and a new virgin male was added at the onset of dusk
on the next day. This protocol was repeated for each female
throughout her life whenever recalling occurred, and her
longevity was recorded. Unreceptive-mated females were
maintained singly because continuous male presence may
significantly enhance remating in laboratory conditions
(Torres-Vila et al., 1997a, 2004), which could result in
overestimation of polyandry. Eggs from the first mating were
checked to ensure that they were fertile. Since once-mated
females laying no fertile eggs unfailingly recall and remate,
these females (, 5%) were excluded from the statistical
analysis to avoid polyandry overestimation. To verify the
number of matings observed and to account for eventually
unobserved ones, all dead females were dissected and the
number of spermatophores counted.
Statistical procedures
Parametric and nonparametric tests were used, depending on
whether the distribution of the response variable could be
normalized. The two estimators of adult size, mid-femur
length (field adults) and body weight (laboratory strain), as
Behavioral Ecology
116
well as female longevity (laboratory strain), were normally
distributed. The effects of sex and larval feeding on adult size
were tested separately for each of the three data sets (field
2000, 2001, and laboratory) in a two-way ANOVA with both
factors treated as fixed effects (Sokal and Rohlf, 1995). A oneway ANOVA was used to test for an effect of larval feeding
treatment on female longevity. Polyandry in L. botrana
(estimated as spermatophores/female) was non-normally
distributed. The effect of adult origin (field 2000, 2001, or
laboratory) and larval feeding on polyandry were therefore
explored in a two-way ANOVA for ranked data according to
the Scheirer-Ray-Hare extension of the Kruskal-Wallis test
(Sokal and Rohlf, 1995). This test was also used to analyze the
effects of year and larval feeding on female reproductive age.
The Spearman rank correlation coefficient (rs) was used to
document relationships between some studied variables
including polyandry, body size, reproductive age, longevity,
and trapping time. Significance of rs values was assessed
through one-tailed tests for small samples (Scherrer, 1984).
Logistic regressions were used to assess the combined effect of
female size and longevity (or reproductive age in field-caught
females) on polyandry, which was coded as a binary dependent variable (0: females mated once, 1: females mated
more than once). The likelihood-ratio test was used to
estimate the effect of each independent variable as well as
their interaction (Sokal and Rohlf, 1995). Most analyses were
computed with Systat (2000) statistical software.
RESULTS
Larval feeding and seasonal variation in polyandry
Adult population dynamics showed a consistent trivoltine
pattern in L. botrana, although a reduced and partial fourth
flight was also apparent in both years (Figure 1). The data
supported the claim that the moth typically undergoes three
complete non-overlapping larval generations in the studied
area. There was considerable temporal variation in the
frequency of polyandry exhibited in the field and it varied
seasonally in a similar way in both years. First-flight (early
spring) females mated more often than did second-flight (late
spring) or third-flight (summer) females (Figures 1 and 2).
The laboratory study confirmed a direct effect of seasonal
changes in larval food supply on polyandry levels (Figure 2).
Polyandry significantly increased from about 1.1 to 1.3
spermatophores per female for larvae reared on inflorescences and ripe grapes, respectively, while those fed on unripe
grapes exhibited an intermediate level of polyandry (Figure 2).
There was a significant effect of larval feeding on subsequent
polyandry levels, while neither adult origin (field or laboratory) nor the origin 3 larval feeding interaction significantly
affected polyandry (Table 1). Note, however, that the p value
from the interaction was marginally nonsignificant (p ¼ .06).
There was a clear trend for an increase in the proportion of
polyandrous females over the course of each flight period
(Figure 1). When the few once-mated females occasionally
captured at the end of some flight periods were excluded
(likely arising due to a protandry-related male deficit, dotted
lines on Figure 1), rs values confirmed a significant increase in
polyandry for all flight periods with sufficient data to compute
rs. L. botrana is a species highly protandrous (Bovey, 1966),
and male shortage later in each flight was especially evident
when examining pheromone trap catches; in all cases, few or
no catches were recorded in these periods (Figure 1).
those reared on unripe grapes or inflorescences (Figure 2). The
number of matings per female tended to increase with female
longevity in all three larval feeding groups (Figure 3), although
the correlation was not significant for females fed unripe
grapes. A similar, but less clear, pattern was found when female
reproductive age was used as an estimator of female chronological age under field conditions. Reproductive age tended to
correlate positively with polyandry but this pattern was in some
instances inconsistent (Figure 3). Female reproductive age at
trapping time was sensitive to environmental conditions, being
significantly affected by year, flight period, and their interaction
(Table 2). Reproductive age had mean values of three scaleunits or higher, indicating that nearly all females had begun egg
laying when they were caught (Figure 1). There was a detectable
increase in female reproductive age over time in some flight
periods, as indicated by rs values (Figure 1). This trend was
consistent with the observed correlation between female
reproductive age and polyandry (Figure 3).
Adult size and polyandry
Our data strongly suggested that the observed differences in
polyandry related to larval feeding were a result of concomitant
changes in adult size. Body size was significantly affected by larval
feeding, both in field-caught and laboratory-tested adults,
irrespective of the size estimator used: body weight or mid-femur
length (Figure 2, Table 3). Confirming previous reports,
individuals that developed on inflorescences were the smallest
and those that developed on ripe grapes were the largest (TorresVila et al., 1999). Females were significantly heavier than males
(Figure 2, Table 3). However, a significant effect of the sex 3
larval feeding interaction on adult weight (Table 3) revealed that
the relative size difference between females and males was
a function of larval feeding (Figure 2). Sexual size dimorphism
(male:female weight ratio) was more pronounced in ripe grape–
reared individuals (4.4:7.7, 57%) than in adults derived from
unripe grapes (3.4:5.6, 61%) or inflorescences (2.7:4.1, 66%). A
less clear picture was observed when mid-femur length was used
as an estimator of adult size in field-caught individuals. Body size
differences between the sexes were significant in 2000 but not in
2001 (Table 3). When the number of spermatophores per female
was plotted against female size (Figure 4), using either midfemur length or body weight, there was a significant positive
correlation between female size and polyandry in all three larval
diets, indicating that larger females had a higher propensity to
remate. There was, however, no significant correlation between
female size and trapping time in any of the six flight periods
studied (all rs , .40, ns). The increase in polyandry within a given
flight period, therefore, cannot be explained by a temporal
increase in female size.
To explore the combined effects of female size and age on
polyandry, we performed logistic regressions. When field data
were analyzed (n ¼ 391 females), female size (G ¼ 3.88, df ¼
1, p , .05), reproductive age (G ¼ 24.23, df ¼ 1, p , .001),
and their interaction (G ¼ 8.37, df ¼ 1, p , .01) all
significantly contributed to explain variation in polyandry.
However, when models were computed with laboratory data
(n ¼ 264 females), only female size significantly affected
polyandry (G ¼ 5.60, df ¼ 1, p , .05), while longevity (G ¼
1.67, df ¼ 1, p ¼ .20) and the size 3 longevity interaction (G ¼
0.06, df ¼ 1, p ¼ .80) did not. These results suggested
a confounding effect of female size on the correlation
between longevity and polyandry shown in Figure 3.
Female age and polyandry
DISCUSSION
Female longevity in the laboratory was significantly affected by
larval feeding. Females reared on ripe grapes lived longer than
L. botrana experiences marked seasonal variation in polyandry
when the moth developed on vine. Polyandry levels follow
Spermatophores
per female
Female
reproductive age
Torres-Vila et al.
•
Polyandry variation in Lobesia botrana
4
7
2
9 20 33
1
6
1
3
29
3
rs=0.50 ns
rs=-0.03 ns
3
27
5 22
4
1 1
6
rs=1.00**
2
1.8
rs=0.98**
1.6
12
2
7
rs=0.98**
1.4
50
1.2
38
54
3
22
20
1 6
1.0
100
Weekly catch
number per
trap
12
117
1 9
3rd flight
2nd flight
1st flight
80
6 4
5
3
1 1
4th flight
60
Pheromone traps
Feeding traps
40
20
0
Spermatophores
per female
Female
reproductive age
Mar
3
4
Jun
Jul
Year 2000
25
Sep
Oct
rs=0.90*
8
16
10
3
22
22
14
29
1
43
1
1
rs=0.88**
2
1.8
1.6
Aug
2
7
7
rs=0.70 ns
2
3
rs=1.00**
rs=0.95*
2
16
rs=1.00*
29 37
1.4
14
1.2
1
1.0
100
Weekly catch
number per
trap
May
Apr
69
25
10
8
1 1
2nd flight
1st flight
80
22
1
3rd flight
4th flight
60
Pheromone traps
Feeding traps
40
20
0
Mar
Apr
May
Jun
Jul
Year 2001
Aug
a cyclic pattern closely related to the quality of food available
for larvae throughout the year. The occurrence of seasonal
variation in polyandry has been documented in a number of
lepidopterans (e.g., Calcote et al., 1984; Howell, 1991;
Watanabe and Ando, 1993), and it has been argued that
seasonal changes in larval food sources may influence female
remating rates (Drummond, 1984). Our results suggest at
least two possible proximate ways in which larval nutrition can
influence the observed level of polyandry in a population.
Female age and polyandry
Female longevity may depend on larval feeding, and longerlived females have more mating opportunities. In L. botrana
the lower polyandry level exhibited by females fed on
Sep
Oct
Figure 1
Seasonal variation in mean (6
SE) female reproductive age
and polyandry (spermatophores per female) in Lobesia
botrana females collected in
baited traps in a vineyard at
La Orden (Spain) during two
consecutive years. Population
dynamics estimated from pheromone traps (males) and
baited traps (males þ females)
are also plotted. Significance
of seasonal increase in female
reproductive age and polyandry within flights was tested
with the Spearman correlation
coefficient (rs). Significance
levels are ***p , .001, **p ,
.01, *p , .05, ns: not significant. Data points connected
with dotted lines were excluded in rs computations (see
text). A small number of data
points (n , 4) precluded rs
computation for polyandry in
the third flight of the year
2000. The fourth flight was
partial both years as is usual
in southern Spain. No catches
occurred in baited traps in the
year 2001 fourth flight. Numbers above data points are
female sample sizes.
inflorescences and unripe grapes could simply be a consequence of their shorter lifespan (6.0 and 7.1 days, respectively) in comparison with females derived from ripe
grapes (13.4 days). Polyandry was significantly correlated with
longevity in two of the three larval food diets (inflorescences
and ripe grapes). However, the presumed positive effect of
female longevity on polyandry must be interpreted with
caution, as it does not necessarily implicate a cause-effect
relationship. Females living longer could have more mating
opportunities, but females mating more often could also live
longer as a result of male nuptial gifts transferred at mating.
Our results evidenced this complex scenario and showed the
potential confounding effect of female size. When logistic
regressions were performed, female longevity did not have an
effect on polyandry after controlling for female size. Thus, at
Behavioral Ecology
118
Spermatophores per female
1.5
1.4
Field (year 2000)
Field (year 2001)
Laboratory strain
1.2
54
69
1.3
86
28
75
78
Source
Sum of squares df
Origin (O)
2960.72
Larval
feeding (L)
475107.69
O3L
76539.60
Error
14236400.00
Total
14791008.01
137
100
1.1
Table 1
Effects of adult origin and larval feeding regime on Lobesia botrana
polyandry estimated as the number of spermatophores per female
123
Mean squares H
2
1480.36
2
4
741
749
237553.85
19134.90
19212.42
19747.67
Hadj
0.15 0.35
p
.84 ns
24.06 56.92 ,.001
3.88 9.17 .06 ns
1.0
Female longevity (days)
16
86
14
12
10
8
78
100
6
4
2
0
10
Adult body weight (mg)
Two-way ANOVA for ranked data according to the Scheirer-Ray-Hare
extension of the Kruskal-Wallis test. Origin includes data from fieldcaught females in 2000 and 2001 and from the laboratory strain
(3 levels). Larval feeding includes data from females developed in
larval stage on inflorescences and unripe and ripe grapes (3 levels).
H statistic is computed as H ¼ SS/MStotal and Hadj is the H value
corrected for ties computed as Hadj ¼ H/D, with D ¼ 0.4227
(Sokal and Rohlf, 1995).
9
8
Females
Males
86
7
78
6
5
100
4
82
3
2
81
88
1
0
Inflorescences
(2nd)
Unripe grapes
(3rd)
Ripe grapes
(1st)
Larval food (adult flight)
Figure 2
Effect of the phenological stage of vine determining feeding
substrate for Lobesia botrana larvae on polyandry (spermatophores per
female), female longevity (days), and adult body weight (mg)
(means 6 SE). Polyandry data sets are from field-caught females
(years 2000 and 2001) and from the laboratory strain reared in larval
stage on vine (see text). Female longevity and adult weight data sets
refer to the laboratory strain. Adult flight order, in connection with
the food available for larvae in each case, is also indicated. Note that
the first flight comprises diapaused adults that fed on ripe grapes the
previous year. Numbers near data points are sample sizes. Female
longevity was significantly different among treatments (ANOVA,
F2,261 ¼ 177.07, p , .001). See Tables 1 and 3 for statistical analysis
on spermatophore number and adult weight, respectively.
least in short-living species such as L. botrana, a causal
connection between longevity and remating in females is
not clear-cut. Biological observations in this moth species also
predict this view. Most polyandrous females (. 90%) only
remated during the few days (4–5 days) after their first
mating, irrespective of larval feeding, and this time period is
shorter than the average longevity of the shortest-lived
females (those developed on inflorescences).
A different picture emerged when female reproductive age
in the field was used in lieu of longevity as an estimator of
female chronological age to explain polyandry. Reproductive
age appeared to be a better estimator of polyandry,
irrespective of larval feeding regime. A logistic regression
showed a significant effect of reproductive age on female
remating when adjusted for female size, despite the occurrence of reproductive age 3 female size interaction. The
possible reasons for a different effect on polyandry of female
age depending on the estimator used to measure it (longevity
or reproductive age) are difficult to interpret and merit
further research.
Significant differences in female reproductive age at
trapping time occurred between years, as well as among flight
periods within a given year. Such differences are attributable
to the variable weather conditions characterizing Mediterranean areas. Summer flying females were, on average, slightly
younger at trapping time than spring flying females. This was
probably a consequence of the improved emission of volatile
compounds by bait traps at high temperatures, and in
summer adults would have a greater attraction to the traps
because of their increased water requirements, to compensate
for transpiration and metabolic losses. Seasonal differences in
reproductive age at capture may also indicate changes in the
relative importance of feeding and reproductive stimuli to
females. The seasonal differences in capture bias did not,
however, substantially alter polyandry estimates under field
conditions, as these closely fit the laboratory data.
A problem often encountered by those using field estimates
of polyandry frequency is that spermatophore counts depend
on the population age structure at the time of sampling, so
field data tends to underestimate actual polyandry levels
(Drummond, 1984; Eberhard, 1985; Pliske, 1973). However, it
has recently been argued that such an underestimation is
likely to be unimportant in monandrous species (Simmons,
2001). Regardless of this criticism, in our study most trapped
females were of fairly advanced age, and spermatophore
counts appear to provide an accurate estimation of polyandry
in the wild, as females had sufficient time to express their
mating potential before being captured.
Adult size and polyandry
Results clearly showed that larval nutrition affected female
size and also suggested that larval feeding background may
affect sexual size dimorphism. The main point is that these
closely related variables may potentially affect in turn levels of
polyandry: the larger the average female size (or the lower the
Torres-Vila et al.
•
Polyandry variation in Lobesia botrana
119
a
2.0
b
1st flight
1.8
ripe grapes
rs=0.60**
2001 (rs=0.55 ns)
2000 (rs=0.89*)
1.6
1.4
10
9
12
5
3
8
15
14
4
9
6
9
3
1.2
9
Spermatophores per female
1.0
2
2.0
20
2227
2
4
22 22
3
1
2nd flight
7
1.8
2001 (rs=0.52 ns)
2000 (rs=-0.50 ns)
1.6
inflorescences
rs=0.96**
8
1.4
54
33
1.2
16
23
28
1.0
2
14
2.0
21
35
16
3rd flight
1.8
3
2
2001 (rs=0.10 ns)
2000 (rs=0.99**)
1.6
43
9
8
14
14
unripe grapes
rs=0.71 ns
14
1.4
19
8
1.2
1
1.0
1
1
12
1.5
2
17
19
24
2.5
3
3.5
Reproductive age (F&L scale)
5
8 26
4
5
10
15
Longevity (days)
male:female size ratio), the greater the polyandry level will be.
Such a relationship may occur in L. botrana because, on the
one hand, the size of spermatophores transferred by males is
positively linked with male size, and on the other, the size of
the female reproductive tract is positively correlated with
female size (Torres-Vila et al., 1995, 1997a, 1999). If female
size increases in a given ecological context (e.g., as a result of
larval feeding) the relative ‘‘filling’’ of females by male
ejaculates may diminish and polyandry will be enhanced
accordingly. This cause-effect relationship occurs through
three well-documented physiological pathways triggering
female post-copulatory refractoriness: (1) the amount of
transferred sperm, (2) the amount of male accessory
glandular substances, and (3) the pressure on nerve receptors
in the bursa copulatrix (Simmons, 2001; Torres-Vila et al.,
1997a and references therein). Leimar et al. (1994) evidenced
how variation in larval food quality may alter relative
male:female size. When food quality for larvae of the butterfly
Pieris napi L. deteriorates, female size decreased more than
male size so that male:female sexual size dimorphism was less
pronounced, i.e., the same pattern observed in L. botrana
(Figure 2). However, a generalization of the above scenario
20
Figure 3
(a) The relationship between
reproductive age (Feldhedge
and Louis scale, see text) and
mean polyandry (spermatophores per female 6 SE) in
Lobesia botrana depending on
the adult flight through the
season. Females were collected
in baited traps in a vineyard at
La Orden (Spain) during two
consecutive years (2000 and
2001). (b) The relationship
between female longevity and
mean polyandry (spermatophores per female 6 SE)
depending on larval feeding.
Data sets are from the laboratory strain reared in larval
stage on vine (see text). rs:
Spearman correlation coefficient. Significance levels are
**p , .01, *p , .05, ns: not
significant. Numbers near data
points are female sample sizes.
seems to be difficult across the Lepidoptera, as a correlation
between female size and polyandry has been documented in
some studies (Bergström et al., 2002; Miyahara, 1978; TorresVila et al., 1997a) but not in others (Bergström and Wiklund,
2002; Svärd and McNeil, 1994; Wedell et al., 2002), even when
carried out on the same target species.
Our results suggested that the impact of larval feeding on
sexual size dimorphism (as well as the occurrence of sex 3
larval feeding interaction) was only consistent when adult
weight was used as an estimator of adult size. When mid-femur
length was used our results were less conclusive. This
disagreement could be a result of the fact that the relative
body-size difference between the sexes depends on the
measurement used (e.g., Rutowski, 1997), or, in other works,
is an outcome of the static allometry characterizing insect
growth (Garcı́a-Barros, 1999; Nijhout, 1994). Lack of consistency between mid-femur-length and body-weight data sets
could be explained by the positive, but variable, scaling
between body size and abdomen size in the Lepidoptera
(Wickman and Karlsson, 1989). When the allometric scaling
relationship y ¼ axb between mid-femur length (y) and body
weight (x) is fitted to both sexes of L. botrana, b values are
Behavioral Ecology
120
Table 2
Effects of year and adult flight on reproductive age of Lobesia
botrana females when they were caught in baited traps
Source
Sum of squares df
Year (Y)
216135.03
Flight (F) 519456.64
Y3F
815355.53
Error
2972199.16
Total
4523146.36
Mean squares H
1 216135.03
2 259728.32
2 407677.77
385
7719.99
390 11597.81
Hadj
p
18.64 19.63 ,.001
44.79 47.17 ,.001
70.30 74.03 ,.001
Two-way ANOVA for ranked data according to the Scheirer-RayHare extension of the Kruskal-Wallis test. Year includes data from
field-caught females in 2000 and 2001 (2 levels). Flight includes
data from the three typical annual flights corresponding to adults
developed when larvae on inflorescences (second flight), unripe
grapes (third flight), and ripe grapes (first flight, diapaused
individuals) (3 levels). H statistic is computed as H ¼ SS/MStotal and
Hadj is the H value corrected for ties computed as Hadj ¼ H/D,
with D ¼ 0.9496 (Sokal and Rohlf, 1995).
consistently lower in females than in males. This fact supports
a proportionally higher increase of abdomen size in females
than in males with body size increase, probably as a consequence of investment in ovarian mass (Torres-Vila LM,
unpublished data).
Patterns from the laboratory and field trials relating larval
feeding, female size, and polyandry were quite consistent, but
it is necessary to note that two uncontrolled effects must be
considered when comparing both data sets. First, mating
opportunities of males were different. The experimental
procedure in the laboratory precluded male remating, as
males were allowed to mate only once, whereas under field
conditions male remating potential was unknown. A higher
larval food quality (ripe grapes) promoting higher size in firstflight L. botrana males could enhance their remating potential
and potentially polyandry levels, as has been shown in
butterflies (Svärd and Wiklund, 1989). However, this notion
is not supported by our results, and moreover, it is unlikely in
a night-flyer moth in which mating is pheromone-mediated
and female-controlled as in L. botrana. Second, the developmental pathway experienced by individuals developed
on ripe grapes was different in the field and laboratory.
Overwintering individuals in the field always experienced
a high quality diet, whereas in the laboratory only directly
developed individuals were used. Pupal diapause might affect
female longevity and pupal weight loss during winter in L.
botrana (Torres-Vila et al., 1996), but the impact of overwintering on female remating, if any, was considered unimportant given the consistent polyandry values obtained in
both trials.
Regardless of all these technical points, our results clearly
support the suggestion that the effect of larval food on female
size can account for much of the seasonal variation in
polyandry in L. botrana. A more subtle effect of sexual size
dimorphism on polyandry is also suggested by our results, but
its true effect (independent of female size) requires additional research.
Temporal increase in polyandry within flight periods
Levels of polyandry increased within each flight period. This
type of trend is often interpreted to be a result of changes in
population age structure, with older females having had more
lifetime mating opportunities (Forsberg and Wiklund, 1989;
Pliske, 1973). Our results support this view, as polyandry was
related to female reproductive age but not to a temporal
increase in female size. However, weak correlations between
female age and polyandry in some flight periods suggest that
additional factors also account for the pattern in L. botrana.
Female remating in this species is a heritable trait (Torres-Vila
et al., 2002), so it would be interesting to know whether there
is a genetic correlation between polyandry and other developmental variables. For instance, if polyandrous females
emerge later after overwintering (Kawagoe et al., 2001) and/
or have slower larval development than monandrous females
(Torres-Vila LM, unpublished data), this would lead to an
increase in polyandry later in each flight period.
Conclusion
Vine phenology, because it affects larval feeding, promotes
a cyclic seasonal pattern of polyandry in L. botrana. The main
proximate factor contributing to individual variation in
polyandry among females is body size, although the effect of
female reproductive age at trapping time must be also
considered. Larval nutrition must therefore be included
among the ecological variables that shape female remating
in this moth. The impact of larval feeding in this species not
Table 3
Effects of sex and larval feeding (related to adult flight in field-caught individuals) on Lobesia botrana adult size (estimated as mid-femur
length or adult weight)
Variable
Source
Sum of squares
df
Mean squares
Mid-femur length (mm)
[2000 field data]
Sex (S)
Larval feeding (L)
S 3 L interaction
Error
Sex (S)
Larval feeding (L)
S 3 L interaction
Error
Sex (S)
Larval feeding (L)
S 3 L interaction
Error
0.119039
0.072911
0.007276
1.867319
0.022379
0.410529
0.012484
2.963634
663.13
629.86
81.16
388.01
1
2
2
280
1
2
2
265
1
2
2
509
0.119039
0.036455
0.003638
0.006669
0.022379
0.205264
0.006242
0.011184
663.13
314.93
40.58
0.7623
Mid-femur length (mm)
[2001 field data]
Adult weight (mg)
[laboratory strain]
F
p
17.85
5.47
0.55
,.001
,.01
.58 ns
2.00
18.35
0.56
.16 ns
,.001
.57 ns
869.91
413.13
53.23
,.001
,.001
,.001
Two-way Model I ANOVAs. Mid-femur length data are from field-caught adults (years 2000 and 2001) whereas adult weight data are from the
laboratory strain reared in larval stage on vine (see text). ANOVAs include data from both sexes (2 levels) developed in larval stage on
inflorescences and unripe and ripe grapes (3 levels).
Torres-Vila et al.
•
Polyandry variation in Lobesia botrana
121
a
2.0
b
1st flight, rs=0.83**
1.8
3
1.6
1
ripe grapes
rs=0.85*
3
29
13
1.4
13
6
7
19
9
10
1.2
9
23
18
1
4
Spermatophores per female
1.0
2.0
1.8
1
inflorescences
rs=0.86**
2nd flight, rs=0.89***
7
1.6
3
13
18
1.4
25
31 39
1.2
7
6
8
35
35 37
3
7 17
1.0
2.0
1.8
5
20
6
38
21
8
23
8
21
19
10
1.2
2
6
7
1.6
1.4
1
unripe grapes
rs=0.94**
3rd flight, rs=0.66*
2
1.0
1.0
1.1
1.2
1.3
1.4
1.5
Mid-femur length (mm)
1.6
1
2
3
4
5
6
7
8
9 10 11
Body weight (mg)
only on polyandry (this study) but also on reproductive
output and the occurrence of heritable variation for polyandry lead us to argue that larval feeding is a key factor with
significant implications for the evolution of polyandry, sperm
competition, and female reproductive strategies.
We thank D. González, L. M. Robredo, D. Uriarte, E. Palo, and A.
Galán for technical assistance in the field or the laboratory. We also
sincerely thank Michael D. Jennions, Christer Wiklund, and one
anonymous referee for helpful comments. Financial and material
support was provided by the Servicio de Investigación y Desarrollo
Tecnológico (SIA) and the Servicio de Sanidad Vegetal, Consejerı́a de
Agricultura y Medio Ambiente, Junta de Extremadura, Spain.
REFERENCES
Arnqvist G, Nilsson T, 2000. The evolution of polyandry: multiple
mating and female fitness in insects. Anim. Behav. 60:145–164.
Bergström J, Wiklund C, 2002. Effects of size and nuptial gifts on
butterfly reproduction: can females compensate for a smaller size
through male-derived nutrients? Behav Ecol Sociobiol 52:296–302.
Bergström J, Wiklund C, Kaitala A, 2002. Natural variation in female
mating frequency in a polyandrous butterfly: effects of size and age.
Anim Behav 64:49–54.
Figure 4
(a) The relationship between
female size (estimated as midfemur length) and mean polyandry (spermatophores per
female 6 SE) in Lobesia botrana
depending on the adult flight
through the season. Females
were collected in baited traps
in a vineyard at La Orden
(Spain) during two consecutive years (2000 and 2001, data
pooled). (b) The relationship
between female size (estimated
as body weight) and mean
polyandry (6 SE) depending
on larval feeding. Data sets are
from the laboratory strain
reared in larval stage on vine
(see text). rs: Spearman correlation coefficient. Significance
levels are ***p , .001, **p ,
.01, *p , .05. Numbers near
data points are female sample
sizes.
Birkhead TR, Møller AP, 1998. Sperm competition and sexual
selection. San Diego, California: Academic Press.
Bovey P, 1966. Superfamille des Tortricoidea. In: Entomologie
appliquée à l’agriculture, 2 (1) (Balachowsky AS, ed). Paris: Masson
et Cie; 859–887.
Calcote VR, Smith JS, Hyder DE, 1984. Pecan nut casebearer
(Lepidoptera: Pyralidae): seasonal activity and mating frequency
in central Texas. Environ Entomol 13:196–201.
Choe JC, Crespi BJ, 1997. The evolution of mating systems in insects
and arachnids. Cambridge: Cambridge University Press.
Coscollá R, 1997. La polilla del racimo de la vid (Lobesia botrana Den. y
Schiff.). Paterna, Valencia, Spain: Generalitat Valenciana, Conselleria de Agricultura, Pesca y Alimentación.
Drummond BA, 1984. Multiple mating and sperm competition in the
Lepidoptera. In: Sperm competition and the evolution of animal
mating systems (Smith RL, ed). New York: Academic Press; 291–370.
Eberhard WG, 1985. Sexual selection and animal genitalia. Cambridge, Massachusetts: Harvard University Press.
Eberhard WG, 1996. Female control: sexual selection by cryptic
female choice. Princeton, New Jersey: Princeton University Press.
Eichhorn KW, Lorenz DH, 1977. Phönologische entwicklungsstadien
der rebe. Nachrichtenbl Dtsch Pflanzenschutzd (Braunschweig) 29:
119–120.
Forsberg J, Wiklund C, 1989. Mating in the afternoon: time-saving in
courtship and remating by females of a polyandrous butterfly Pieris
napi L. Behav Ecol Sociobiol 25:349–356.
122
Garcı́a-Barros E, 1999. Implicaciones ecológicas y evolutivas del
tamaño en los artrópodos. Bol SEA 26:657–678.
Howell JF, 1991. Reproductive biology. In: Tortricids pests, their
biology, natural enemies and control (Van der Geest LPS, Evenhuis
HH, eds). Amsterdam: Elsevier; 157–174.
Jennions MD, Petrie M, 2000. Why do females mate multiply? A review
of the genetic benefits. Biol Rev 75:21–64.
Kawagoe T, Suzuki N, Matsumoto K, 2001. Multiple mating reduces
longevity of females of the windmill butterfly Atrophaneura alcinous.
Ecol Entomol 26:258–262.
Leimar O, Karlsson B, Wiklund C, 1994. Unpredictable food and
sexual size dimorphism in insects. Proc R Soc London B 258:
121–125.
Marshall LD, McNeil JN, 1989. Spermatophore mass as an estimate of
male nutrient investment: a closer look in Pseudaletia unipuncta
Haworth (Lepidoptera: Noctuidae). Funct Ecol 3:605–612.
Miyahara Y, 1978. Effect of the intake of sugar at adult stage on the
mating of Leucania separata and Agrotis ipsilon. Proc Assoc Plant Prot
Kyushu 24:101–104.
Nijhout HF, 1994. Insect hormones. Princeton, New Jersey: Princeton
University Press.
Pliske TE, 1973. Factors determining mating frequencies in some new
world butterflies and skippers. Ann Entomol Soc Amer 66:164–169.
Ridley M, 1988. Mating frequency and fecundity in insects. Biol Rev
63:509–549.
Rutowski RL, 1997. Sexual dimorphism, mating systems and ecology
in butterflies. In: Mating systems in insects and arachnids (Choe JC,
Crespi BJ, eds). Cambridge: Cambridge University Press; 257–272.
Scherrer B, 1984. Biostatistique. Québec, Canada: Gaëtan Morin.
Simmons LW, 2001. Sperm competition and its evolutionary consequences in the insects. Princeton, New Jersey: Princeton University Press.
Simmons LW, 2003. The evolution of polyandry: patterns of
genotypic variation in female mating frequency, male fertilization
success and a test of the sexy-sperm hypothesis. J Evol Biol 16:
624–634.
Sokal RR, Rohlf FJ, 1995. Biometry: the principles and practice of
statistics in biological research, 3rd ed. New York: Freeman.
Stockel J, Roehrich R, Carles JP, Nadaud A, 1989. Technique d’élevage
pour l’obtention programmée d’adultes vierges d’Eudémis. Phytoma 412:45–47.
Svärd L, McNeil JN, 1994. Female benefit, male risk: polyandry in
the true armyworm Pseudaletia unipuncta. Behav Ecol Sociobiol 35:
319–326.
Svärd L, Wiklund C, 1989. Mass and production rate of ejaculates in
relation to monandry/polyandry in butterflies. Behav Ecol Sociobiol 24:395–402.
Systat, 2000. SYSTAT 10.0. The system for statistics. Richmond,
California: Systat Software Inc.
Thornhill R, Alcock J, 1983. The evolution of insect mating systems.
Cambridge, Massachusetts: Harvard University Press.
Behavioral Ecology
Torres-Vila LM, 1996. Efecto de la temperatura de desarrollo
preimaginal sobre el potencial biótico de la polilla del racimo de
la vid, Lobesia botrana (Denis y Schiffermüller, [1775]) (Lepidoptera:
Tortricidae). SHILAP Revta Lepid 24:197–206.
Torres-Vila LM, 2000. Lobesia botrana Den. and Schiff. (Lepidoptera:
Tortricidae) datasheet. In: Crop protection compendium, 2nd ed
(CAB-International, ed). Wallingford-Oxon, England: Commonwealth Agricultural Bureau-International; CD-ROM.
Torres-Vila LM, Gragera J, Rodrı́guez-Molina MC, Stockel J, 2002.
Heritable variation for female remating in Lobesia botrana, a usually
monandrous moth. Anim Behav 64:899–907.
Torres-Vila LM, Rodrı́guez-Molina MC, 2002. Egg size variation and its
relationship with larval performance in the Lepidoptera: the case of
the European grapevine moth Lobesia botrana. Oikos 99:272–283.
Torres-Vila LM, Rodrı́guez-Molina MC, Gragera J, Bielza-Lino P, 2001.
Polyandry in Lepidoptera: a heritable trait in Spodoptera exigua
Hübner. Heredity 86:177–183.
Torres-Vila LM, Rodrı́guez-Molina MC, Jennions MD, 2004. Polyandry
and fecundity in the Lepidoptera: can methodological and conceptual approaches bias outcomes? Behav Ecol Sociobiol 55:315–324.
Torres-Vila LM, Rodrı́guez-Molina MC, Roehrich R, Stockel J, 1999.
Vine phenological stage during larval feeding affects male and
female reproductive output of Lobesia botrana (Lepidoptera:
Tortricidae). Bull Entomol Res 89:549–556.
Torres-Vila LM, Stockel J, Bielza P, Lacasa A, 1996. Efecto de la
diapausa y del capullo sobre el potencial biótico de la polilla del
racimo Lobesia botrana Den. y Schiff. (Lepidoptera: Tortricidae). Bol
San Veg Plagas 22:27–36.
Torres-Vila LM, Stockel J, Rodrı́guez-Molina MC, 1997a. Physiological
factors regulating polyandry in Lobesia botrana (Lepidoptera:
Tortricidae). Physiol Entomol 22:387–393.
Torres-Vila LM, Stockel J, Roehrich R, 1995. Le potentiel reproducteur et ses variables biotiques associées chez le mâle de l’Eudémis
de la vigne Lobesia botrana. Entomol Exp Appl 77:105–119.
Torres-Vila LM, Stockel J, Roehrich R, Rodrı́guez-Molina MC, 1997b.
The relation between dispersal and survival of Lobesia botrana larvae
and their density in vine inflorescences. Entomol Exp Appl 84:
109–114.
Watanabe M, Ando S, 1993. Influence of mating frequency on lifetime
fecundity in wild females of the small white Pieris rapae (Lepidoptera: Pieridae). Jpn J Entomol 61:691–696.
Wedell N, Wiklund C, Cook PA, 2002. Monandry and polyandry as
alternative lifestyles in a butterfly. Behav Ecol 13:450–455.
Wickman PO, Karlsson B, 1989. Abdomen size, body size, and the
reproductive effort of insects. Oikos 56:209–214.
Yasui Y, 1998. The ‘‘genetic benefits’’ of female multiple mating
reconsidered. Trends Ecol Evol 13:246–250.
Zeh JA, Zeh DW, 2001. Reproductive mode and the genetic benefits of
polyandry. Anim Behav 61:1051–1063.
Zeh JA, Zeh DW, 2003. Toward a new sexual selection paradigm:
polyandry, conflict and incompatibility. Ethology 109:929–950.