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INTRODUCTION
Phenotypic plasticity in insect life histories provides a
way of optimising phenotype to the environment, which
may substantially contribute to individual fitness in
seasonal habitats (Tauber & Tauber, 1981;
West-Eberhart, 1989; Nylin & Gotthard, 1998). Recent
studies on plastic responses in the phenology of butterfly
life histories have often focused on three crucial traits, i.e.
size, development time and growth rate (Nylin, 1994).
Pupal weight is often taken as a surrogate of adult body
weight (e.g. Wiklund & Solbreck, 1982; Nylin et al.,
1993; Soontiëns & Bink, 1997; Kemp, 2000). In addition,
pupal weight is also an accurate expression of the total
weight gain achieved by the larvae. This “composite”
interpretation of the role of the pupal stage may mask
some complex underlying relations, such as sex-linked
differential investment of larval-gathered resources into
adult structures (the more evident example of this being
sexually dimorphic adult to pupal weight ratios). A
further insight into plastic responses during the larval
stage may be gained by using measurements of larval size
that are independent of weight and show a variable
response to environmental factors.
Head capsule width is one such trait because it provides
a linear measurement of larval size that can be contrasted
with pupal or adult sizes. In Lepidoptera (and other holo-
metabolous insects with eruciform larvae), the larval head
capsule is the most conspicuous fully sclerotized
structure. Head capsule growth is basically restricted to
the period of ecdysis and the head sizes of successive
larval instars tend to follow a regular (often exponential)
progression (Dyar, 1890). This regularity has been for-
malised in a number of ways and widely applied to identi-
fying larval instars (e.g. Gargiullo & Berisford, 1982;
Fink, 1984). Such use is not efficient in all instances due
to overlapping frequency distributions of the larval head
widths of successive instars and variable number of
instars (Gaines & Campbell, 1935; Goettel & Philogene,
1979). The causes of the variation in number of instars
include genetic variation (e.g. Zhou & Topp, 2000), dia-
pause (Yin & Chippendale, 1974), compensatory growth
on poor quality substrates, sexual dimorphism, tempera-
ture, growth rates or even larval gregariousness (reviewed
by Wigglesworth, 1972). Thus, variability in the number
of larval instars may represent a plastic response to envi-
ronmental factors, and provide a tool for identifying dif-
ferent potential developmental pathways, their causes and
effects on adult size. Three points are of interest here:
firstly, do additional larval instars result in larger final
instar larvae (with a potential positive effect on pupal
size), or just compensate for poor growth in early instars.
Secondly, do the larger final instar larvae develop into
larger pupae, or adults. Thirdly, to what extent do these
effects differ between populations, sexes or generations
and indicate different patterns of allocation of larval
resources into adult soma.
One comparison of this kind was possible using mate-
rial derived from a wider rearing experiment of a Medi-
terranean population of the small heath butterfly,
Coenonympha pamphilus (L., 1758). This satyrine Nym-
phalid is widespread in the Western Palaearctic (Kudrna,
2002). The larvae feed on several grass species, the adults
are on the wing in spring and summer, and produce one
or more broods depending on latitude and altitude. The
larvae of this species are reported to moult three times,
hence undergoing four larval instars, and to hibernate in
diapause in the third (penultimate) instar (Roos, 1978;
Eur. J. Entomol. 103: 47–53, 2006
ISSN 1210-5759
Number of larval instars and sex-specific plasticity in the development of the
small heath butterfly, Coenonympha pamphilus (Lepidoptera: Nymphalidae)
ENRIQUE GARCÍA-BARROS
Department of Biology (Zoology), Universidad Autónoma de Madrid, E-28049 Madrid, Spain; e-mail: garcia.barros@uam.es
Key words. Lepidoptera, butterfly, Satyrinae, Coenonympha, development, diapause, growth, head capsule, instar, moulting,
phenotypic plasticity, seasonality
Abstract. The number of larval moults, larval head capsule width and pupal weight were investigated in both direct-developing and
diapausing individuals of a South-West European population of Coenonympha pamphilus. The frequency distributions of head
widths of successive larval instars overlapped, partly due to variation in the number of larval moults. The larvae that entered dia-
pause went through five instars, instead of the four reported from this species. The evidence indicates that the five instar develop-
mental pathway represents a plastic response rather than an example of compensatory growth. This alternative growth pattern was
expressed in response to short photoperiods in parallel with, or as a consequence of, larval diapause. On average, the larvae with five
instars had larger heads than their normal siblings. This resulted in comparatively heavier male pupae, while the opposite trend
occurred in females. It is concluded that the variation in the number of larval instars is a plastic response to diapause when tempera-
tures remain mild and that it might have an adaptive value in areas with mild winter climates. The sexually dimorphic expression in
the larval growth patterns, in terms of pupal weight, may well imply different patterns of allocation of larval resources to adult struc-
tures, although sex-dependent differences in investment into purely larval structures cannot be discounted.
47
Wickman et al., 1990). In a study of the larval phenology
of this insect in Central Spain, a degree of heterogeneity
in the weight of diapausing larvae was detected, throwing
some doubt on the identity of the hibernating instar.
Detailed data on the head widths and pupal weights of a
number of individuals were recorded during this study, of
both direct developing and diapausing larvae. This data
proved interestingly that the number of instars is poten-
tially variable in this species. Given this variation, the
current study had two objectives. First, to determine the
extent to which the presence of an extra instar was deter-
mined or induced by larval diapause. Second, and more
interesting, to document whether the “one extra instar”
developmental pathway represented the expression of a
reaction norm involving sexually dimorphic patterns in
the allocation of larval somatic materials in the pupae.
MATERIALS AND METHODS
Eggs were obtained from females collected at Manzanares el
Real (province of Madrid, Spain: 40°43´N, 4°08´W, 900 m
a.s.l.). The eggs were kept under standard conditions (tempera-
ture of 23 ± 0.8°C, photoperiod of 16L : 8D) until eclosion. The
newly-hatched larvae were transferred to individual plastic jars
(250 ml) containing a small amount of substrate (ca. 50 cm3 of
garden soil) with grass growing in it. A commercial grass mix-
ture of Festuca rubra and Poa annua was used as food. The
grass was planted in large pots and transferred to the jars when
it was ca. 10 cm height. The larvae were regularly transferred to
new jars with fresh grass, and inspected periodically.
To measure the head width, the larvae were gently placed on
a piece of filter paper in the bottom of a Petri dish softly illumi-
nated. As soon as the larvae ceased moving, the maximum head
width was measured from above. This was done to the nearest
division of a micrometric grid incorporated in a binocular
microscope (1 division = 0.035 mm). Additional head widths of
larvae of unknown instar were measured at other times in this
study. Measuring the head capsules of living larvae made it pos-
sible to compare the linear measurements of all the larval
instars: the head capsule widths of the last instar larvae cannot
be measured using exuviae and those of the smallest larvae were
often lost in the rearing jars. Fortunately, the larvae of this spe-
cies are slow-moving, and can easily be managed in the way
described above. A measurement error of at most 1–3 scale units
(equivalent to 0.11 mm) is assumed for the largest head capsules
(this was not determined specifically). The pupae were weighed
to the nearest 0.01 mg within 24 h of pupation.
Collection data did not conform to an experimental design,
since a variable number of instars was not a priori expected.
Instead, the study is based on subsets of larvae that were col-
lected and measured as described above. Fortunately, however,
these included several subsets of larvae derived from the same
females but reared under different conditions. Given that at most
five eggs laid by each female were included in the same set and
that the initial sizes of these were 40–50 eggs, the larval groups
were treated as randomly selected and therefore any potential
maternal effects can reasonably be ignored.
The larvae were reared either outdoors (natural temperature
and photoperiod), or under controlled conditions (fixed photope-
riod and temperature). Both rearing protocols were such that
some part of the larvae exhibited diapause and others developed
directly. Outdoor rearing was carried out on the Campus of the
Universidad Autónoma de Madrid, and included larvae from
eggs hatched during late April and early May (of which all the
larvae developed into adults in summer), and larvae born in the
first half of August, which entered winter diapause and with few
exceptions pupated the next spring. Controlled laboratory
rearing was done in environmental cabinets at a constant tem-
perature of 23°C (±0.8°C) constant temperature, and photope-
riods of 16L : 8D (direct development), 14L : 10D and 13L :
11D. The majority of the larvae of C. pamphilus from this popu-
lation enter diapause at day lengths of 14 h or less (unpubl.).
Given the relatively small sample sizes, the two last groups
(14L and 11L) were treated as the same sample. Diapausing
larvae were transferred to a 16L : 8D photoperiod after more
than 40 days in diapause in order to induce the completion of
larval development and pupation. Larval diapause continued
under this high temperature (23°C) for as long as would nor-
mally be expected under field conditions in winter. This may be
one reason why some part of the larvae kept in these conditions
failed to pupate, which also reduced sample sizes. Only indi-
viduals that successfully developed to the adult stage were used
for detailed comparisons, although the data from a few more
individuals that pupated but were lost before reaching the adult
stage were incorporated for comparisons where sex was not
involved.
RESULTS
Number of larval instars
The frequency distribution of larval head capsule
widths collected during this study (all available measure-
ments) is shown in Fig. 1. Apart from the neat peak for
the first instar and, to some extent, the second and third
instars, there were no clear-cut discrete size classes (Fig.
48
Fig. 1. Frequency distribution of the head capsule widths of
larval Coenonympha pamphilus reared in the laboratory under
different conditions (number of individuals on the Y axis): a =
all available measurements, b = larvae with four instars, c =
larvae with five instars (head widths of the fourth instar indi-
viduals of this group are shown separately). Note that the
sample size in a is larger than in b + c.
1a). An inspection of the individual growth histories dem-
onstrated that some of the larvae had undergone four
instars, while others underwent one further (fifth) instar.
Even when the two groups were plotted separately (Fig.
1) a degree of overlap between the size ranges of succes-
sive instars remained. In the larvae with five instars, this
was true for the three last instars. There were no signifi-
cant differences between the mean head capsule width of
the first instar of both groups (F1, 401 = 0.22, P = 0.64),
while that of second instar larvae that underwent five
instars was smaller than that of larvae with four instars
(F1, 401= 12.46, P = 0.0005) (Table 1).
The average head width growth relative to instar
number was consistent within each group of larvae (Fig.
2). Larvae with five instars had a lower rate of head
growth relative to instar number, since the slopes of the
regression lines (head width on instar number, Fig. 2)
were significantly different (respectively 0.21 and 0.17;
ANCOVA, F = 322.2, P < 0.0001, d.f. = 1). On average,
the larvae that went through five instars had a larger final
instar head capsule width than the larvae with four instars
(ANOVA, F1, 341 = 12.8, P = 0.0004), even when the dis-
tribution ranges of the two groups in their final instar
were roughly similar and the maximum values were
recorded for larvae that had four instars (Fig. 1). The cor-
relations between instar-specific head widths, as well as
between these and pupal weight for the two groups of
larvae are given in Table 2.
Number of instars, rearing conditions and sex
Fifth instar larvae were detected only among those kept
in conditions promoting diapause (test in the proportions
49
840.2761.867———5
1090.2391.3364980.2421.7044
1170.1060.9284930.1441.0843
1070.0620.6384570.0940.6912
1070.0620.3863050.0310.3821
ns.d.Meanns.d.Mean
Larvae with 5 instarsLarvae with 4 instars
Instar
TABLE 1. Average maximum head widths (in mm) for all the
larvae with four and five larval instars (s.d.= standard deviation,
n = sample size; total n varies between instars due to the addi-
tion of some data for larvae with a known number of instars but
for which not all instars were measured).
Fig. 2. Relationship between mean larval head width and
instar for larvae with four (solid squares, dashed line) and five
instars (empty squares, solid line). Vertical bars represent ±1
standard deviation; head widths were log-transformed. The
regression lines and statistics (calculated using the instar
average in each of the two subsets) are: four instars, Log10 head
width = –0.616 + 0.214 (instar number) (r = 0.998, P = 0.002, n
= 4); five instars, Log10 head width = –0.558 + 0.160 (instar
number) (r = 0.996, P = 0.0002, n = 5).
0.40**L5
0.22(P=0.07)0.29*L4
0.29*0.36**0.56***L3
–0.01(ns)0.17(ns)0.26* 0.50***L2
0.12(ns)0.03(ns)0.04(ns)–0.03(ns)–0.02(ns)L1
Five instars
0.30**—L4
0.11(P=0.07)—0.50***L3
0.02(ns)—0.20** 0.59***L2
–0.04(ns)—0.01(ns)–0.06(ns)–0.12(ns)L1
Four instars
Pupal weightL5L4L3L2
TABLE 2. Relationships between the successive larval head
sizes of individual larvae, and between larval head width and
pupal size, measured as correlations (Pearson r values) for
larvae with four and five instars. Sample sizes as in Table 1. L1
to L5 = first to fifth larval instars. *** = P < 0.001, ** = P <
0.01, * = P < 0.05, ns = not significant (exact values given for
non-significant relations where P is close to 0.05). Note that
there are only significant correlations between successive larval
head widths in the latter instars (the head sizes of the interme-
diate and final instars were largely independent of first instar
larval head widths), and that pupal weight was broadly inde-
pendent of the head sizes of the first two larval instars (and
weakly, or non significantly correlated with the penultimate and
final instar head widths).
Fig. 3. Summary of the relations between the overall rearing
conditions (conditions promoting either direct development or
diapause), actual developmental pathway, number of instars, sex
and sample size (n). Average last instar larval head width, and
pupal weights for males (solid circles) and females (empty cir-
cles) are given together with bars indicating 95% confidence
ranges (n = sample size).
of larvae with five instars in photoperiods equal to or
longer than 15L : 9D vs. those shorter than 15L: 9D, Fig.
3: X2 = 65.22, P < 0.0001, d.f. = 1). If the actual develop-
mental pathway (i.e., diapause or direct development) is
considered, its association with the number of larval
instars is even stronger (comparison of the proportions of
larvae with one extra instar in the direct developing and
diapausing larval groups: X2 = 84.51, P < 0.0001, d.f. = 1,
Fig. 3). In other words, the conditions inducing diapause
also induced the 5-instar development pathway in some
larvae, while five instars were very rarely recorded under
conditions favouring direct development (Fig. 3). Overall,
the number of larval instars was independent of sex: X2=
0.03, P= 0.86, d.f. = 1 (incidentally, the sex ratio
departed slightly from equality, 55% males: 45%
females).
Relationships between number of instars, head
capsule width and pupal weight
Pupal weight was correlated with last instar larval head
width for the whole sample. This relationship was statisti-
cally significant, but not strong (Fig. 4). More detailed
inspection of the data revealed that the correlation is sig-
nificant for males (r = 0.249, P = 0.0002, n = 216), but
not females (r = 0.049, P = 0.52, n = 174). These trends
were confirmed for each of the instar groups within each
sex (Fig. 4). It was also evident that the ratio of pupal
weight to larval head size was higher for female than
male individuals. In spite of the within-group correlations
described above, a comparison of the mean pupal weights
and head sizes between instar groups for each sex (Fig.
50
Fig. 4. Relationship between the head width of the last instar
larva and pupal weight in males (above) and females (below).
For both sexes, individuals with four larval instars are indicated
by solid dots, and those with five instars by open circles. The
dashed line shown in both plots represents the regression line
fitted to the whole sample (males and females pooled): log
(Pupal weight) = 0.64 + 0.71log (Larval head width), r = 0.29 g
P < 0.0001, n = 343. The correlations for each sex and number
of larval instars are: males (four instars), r = 0.19, P = 0.016;
males (five instars), r = 0.58, p < 0.0001; females (four instars),
r = 0.08, P = 0.35; females (five instars), r = 0.20, P = 0.23.
0.0063862.425Error
P < 0.000125.00.15720.314Development (sex)
P < 0.0001283.91.78311.783Sex
FMSdfSS(b)
0.0073852.527Error
P = 0.000110.940.07220.144Instars (sex)
P < 0.0001122.360.80310.803Sex
P = 0.000616.450.10810.108Head width
FMSdfSS(a)
Dependent variable = pupal weight
TABLE 3. Tests for the effects of sex and type of larval devel-
opment (direct or diapause) on pupal weight for all the larvae,
based on log-transformed data. Nested ANCOVA analyses (last
instar larval head width set as covariate) for, (a): Comparison
between male and female pupal weights within each instar class
(4 or 5). (b): Comparison between the pupal weights of direct
developing and diapausing individuals (Development) within
each sex. In both instances the last instar larval head width was
set as covariate. Pupal weight was found to differ between sexes
in each instar class, as well as between the types of development
within each sex.
0.0071781.169Error
P = 0.00956.8730.04510.045Effect
FMSdfSS(b)
0.0071781.169Error
P = 0.02013.9960.02620.052Instars (sex)
P < 0.000187.4740.57410.574Sex
P = 0.000214.2450.09410.094Head width
FMSdfSS(a)
Dependent variable = pupal weight
TABLE 4. Tests of the effects of sex and of the number of
instars on pupal weight within each sex, taking into account the
general effect of last instar head width. The comparison is
restricted to larvae that entered diapause. Above (a), comparison
between the pupal weights of diapausing individuals with 4 or 5
instars (Instars) within each sex (nested ANCOVA, head width
set as covariate). Below (b), test for planned comparison fol-
lowing the main results of (a); the effect tested is that pupal
weights (relative to head width), fit the hypothesis: (PWf5<
PWf4 and PWm5 > PWm4), where PW = pupal weight, f =
female, m = male, and 4–5 is the number of larval instars. The
number of larval instars had a significant effect on pupal weight
within each sex (a), and this effect differed as predicted by the
above hypothesis (b): adding one instar resulted in compara-
tively heavier male pupae and lighter female pupae (both rela-
tive to last instar larval head size).
5a) suggested an overall negative relationship between
these two variables (Table 3a). This resulted because
directly developing individuals of both sexes were
heavier than diapausing ones (Table 3b) and very few of
the direct developing individuals moulted more than three
times (2% at most, Fig. 3). A similar plot of the data of
larvae of known sex that entered diapause suggested that,
within these conditions, the males that had an additional
instar (and hence larger heads) metamorphosed into
heavier pupae, while the opposite was the case for
females (Table 4; Fig. 5b). This is also the case when the
data is grouped according to sex, number of instars, and
rearing conditions (Fig. 6; nested ANCOVA: F1, 174 = 5.07
P = 0.026).
DISCUSSION AND CONCLUSIONS
The results indicate that larval head width is not a reli-
able tool for identifying instar in this butterfly. It has to
be stated, however, that the overlapping in the frequency
distributions of the instar-specific head capsule widths
was to some extent increased by combinating data from
larvae reared under different conditions. If restricted to
larvae reared under the same conditions and showing
direct development, the four instars can usually be readily
identified by eye. As formerly shown for a number of
Lepidoptera and other insects, the variable number of
instars in C. pamphilus is not uncommon (further exam-
ples in Silver, 1958; Kishi, 1971; Schmidt et al., 1977;
Daly, 1985; Weatherby & Hart, 1986; Pershing et al.,
1988). In C. pamphilus, however, an extra instar occurs in
some of the larvae that undergo diapause when tempera-
tures are mild. In these conditions the diapausing larvae
continue to feed. The same explanation applies to larvae
reared under seminatural conditions from eggs hatched in
late summer in this study. They started to feed on pot-
grown fresh grass at a relatively early date compared to
wild individuals, and hence had more time for growth
(probably as much as one month: García-Barros,
unpubl.). Similarly, they experienced decreasing late-
summer photoperiods and the majority of them entered
diapause.
The observations presented, including the correlations
between the head widths of consecutive instars, support
the idea that a fifth larval instar is a potential sub-routine
of growth associated with diapause. Further, the option of
an extra instar appears to be determined early in larval
life, probably before the first moult. This mode of devel-
opment is characterised by a comparatively low rate of
larval size increase relative to instar number, with the
mean larval sizes of successive instars following a regular
progression. The “five-instar” diapausing pathway does
not compensate for poor growth in early instars (because
food plant quality was the same in all experiments), nor is
it related to sex. Thus, it is tempting to speculate on its
possible adaptive significance. From this point of view it
is worth noting that the incidence of five larval instars
51
Fig. 5. Relationship between mean last instar larval head
width and average pupal weight for males (squares) and females
(circles) that underwent four (solid) and five (open) larval
instars. Bars indicate 95% confidence limits for the mean and
numbers sample sizes. Above, all individuals for which there
were head measurements and pupal weights; below, only those
individuals that entered larval diapause. All data were logarith-
mically transformed.
Fig. 6. Mean weights of pupae (log-transformed pupal
weights) of diapausing individuals of C. pamphilus of both
sexes and both instar goups (four and five instars), reared either
under constant temperature and day length conditions or out-
doors. Circles = males, squares = females, black = four instars,
open = five instars. Bars indicate 95% confidence limits.
was low and that the conditions promoting this growth
pattern are unlikely to occur frequently throughout this
insect’s geographic range. Co-occurrence of short photo-
periods inducing diapause and mild temperatures may
predictably occur, however, in the South of Europe. The
“five-instar” slow developing larvae are here hypothe-
sized as a way of taking advantage of available resources
(food plants and temperature) without abandoning larval
diapause (as recorded for other insects: Danks, 1994).
This makes sense in areas like the Mediterranean region,
where the above conditions frequently occur in an unpre-
dictable way during autumn. This is consistent with the
fact that polymorphic insect life cycles are often associ-
ated with habitat unpredictability (Tauber & Tauber, 1981
and references therein).
Adaptive explanations such as that proposed above rely
on the assumption that some additional larval growth may
enhance individual fitness. Since adding one instar
resulted in larger last instar larvae, greater pupal weights
(and hence larger adults) are to be expected for insects of
both sexes. This was confirmed for males, but the reverse
was the case for female pupae of the five-instar group.
This relationship is difficult to explain without reference
to development times or growth rates (e.g. Wiklund et al.,
1991; Nylin, 1992). However, a purely size-based inter-
pretation is pertinent, for we are dealing with the relation-
ship between two estimates of size (i.e. larval head and
pupal size) and two alternative adaptive explanations are
possible. First, and assuming that larval ingestion and
assimilation rates are similar for the larvae of both sexes,
female larvae might invest proportionally more in larval
tissues not destined for conversion into adult structures
(e.g. a tougher cuticle or other features that could enhance
winter larval survival). Second, post-diapause larval
growth, which in nature means spring growth, presents
different demands for the two sexes. Females accumulate
a proportionally higher amount of fat, perhaps incurring
costs in terms of investment in other body structures. This
would make sense if the nutrient assimilation of the dif-
ferent larval instars differed, with the last instar contrib-
uting comparatively little to the reproductive reserves (as
shown for other insects: Flanagin et al., 2000). Thus both
adaptive and constraint-based hypotheses are possible. A
closer investigation of the correlation between instar
number and larval size, as well as between these and
pupal or adult size, are needed for a more thorough inter-
pretation of the observed pattern.
Similar observations from related taxa, or those with a
comparable phenology in seasonal environments, would
also be of interest. In fact, variation in the number of
instars in circumstances similar to those described above
may not be unusual. The author (García-Barros, 1988)
observed a similar type of behaviour in hibernating larvae
of the univoltine satyrine, Hipparchia alcyone (D. &
Schiff., 1775). These larvae were accidentally kept in
mild temperature conditions during winter: some of the
larvae went through a further (sixth) instar and metamor-
phosed into adults that were of a larger size than those
that developed from their five-instar siblings. In a series
of papers on the phenology of the satyrine Pararge
aegeria (L., 1758) in Britain, Tilley (1993–1997) reports
developmental polymorphism that mirrors that described
in this study. In P. aegeria some of the hibernating larvae
go through an extra instar although, unlike C. pamphilus,
these individuals usually hibernate as pupae (Tilley, 1993,
1996). Even when there is evidence of an extra instar
occurring in larvae exposed to short photoperiods (Tilley,
1997), the comparison with C. pamphilus is not straight-
forward: P. aegeria can hibernate as either a pupa or a
larva and there is no data on adult or pupal sizes from
those experiments. Further comparative studies on pheno-
logically similar species with hibernating larvae in areas
with a mild winter season, as well as on taxonomically
allied Lepidoptera, are needed to fully determine the
adaptive nature and possible phylogenetic origin of this
reaction norm.
ACKNOWLEDGEMENTS. I thank T. Esperk and two anony-
mous reviewers for useful comments and advice on the literature
concerning varying number of instars in Lepidoptera and other
insects.
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Received March 17, 2005; revised and accepted July 1, 2005
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