J. Ethod.

9:47 50, 1991

Effects of Spermatophore Size on Female Remating in the Armyworm,

Pseudaletia separata, with Reference to Larval Crowding

Yibin HE and Yoshitaka TSUBAKI 1

Laboratory of Applied Entomology and Nematology, Faculty of Agriculture,
Nagoya University, Nagoya 464, Japan
(1present address: Laboratory of Wildlife Conservation, National Institute for
Environmental Studies, Onogawa, Tsukuba 305, Japan)

Abstract -- Effects of spermatophore size on female remating were studied using the
armyworm, Pseudaletiaseparata Walker, reared under isolated and crowded larval condi-
tions. Females that received large spermatophores remated in lower proportion than
those receiving small ones. Spermatophore size of the first mating was estimated by using
mathematical models by He and Tsubaki (in press). Females reared in isolated conditions
were more likely to remate than those reared in crowded conditions.

The armyworm (Pseudaletia separata, Walker),
one of the worldwide pests of gramineous
crops, shows phase-like polymorphism. Under
low density conditions larvae are pale green
(solitary phase); at high-density conditions they
become blackish (gregarious phase) (Iwao,
1968). In a previous paper (He and Tsubaki, in
press), we reported that the males from larvae
reared in groups produced larger sperma-
tophores than the males from solitary-reared
larvae, despite the fact that adult body weight
was not different between the 2 types.
Several potenstial costs that males incur in
production of spermatophore have been sug-
gested (Dewsbury, 1982): time cost of pro-
longed mating, energy cost to replenish acces-
sory gland materials to form spermatophore,
etc. Conversely, spermatophore materials may
provide energy for spermatozoa (Osanai et al.,
1987), or provide nutrition to females that can
increase quantity and quality of eggs (e.g.,
Boggs and Gilbert, 1979; Watanabe, 1988;
Oberhauser, 1989). Spermatophore size may
also influence the length of the female refrac-
tory period for remating (e.g., Sugawara, 1979;
Oberhauser, 1989), therefore affecting the re-
groduetive output of both males and females.
In this paper, we report results of an experi-
ment examining the effect of spermatophore
size on female remating.
Methods
Insects were reared at a constant temperature
(25 _+ I~ , 16L:8D). The dark photophase was
set from 10:00 to 18:00 for the convenience of
observation. Larvae were reared in plastic cups
(3.5 cm depth X 7.5 cm dia.) and fed on artifi-
cial diet (Insecta LF ~e, Agriculture Production
Industry Limited Co., Japan) until pupation.
About 100 larvae were individually reared in
solitary conditions (1 larva/cup, hereafter
"solitary-type"), and another 100 larvae under a
crowded condition (10 larvae/cup, hereafter
"gregarious-type"). Although quantitative data
are lacking, body colour of the former was
mainly pale green and that of the latter was
dark brown. This colour change is characteristic
of the phase variation in this species (Iwao,
1968). On the morning after adult eclosion
(when adults had dried and were able to fly),
moths were weighed, individually marked on
the wings with a quick-drying color pen (Opa-
que Color, Magic Inc~), and stored in meshed
cages (40 • 40 • 40 cm), supplied with cotton-
48

a b s o r b e d 10% h o n e y solution. Males and Ss 0.404 + 0.062A ( A indicates male age at

females were kept in different cages to prevent
copulation.
T h e mating e x p e r i m e n t was carried out in a
mating cage (40 X 40 x 40 cm) in the same
r o o m as the r e a n n g process. Males and females
were i n t r o d u c e d into these cages, at a sex ratio
of about 1 : 1. The moths were released into the
cage just before the d a r k photophase ( 9 : 3 0 -
10:00), and r e m o v e d after the d a r k p h o t o p h a s e
( 1 8 : 0 0 - 1 9 : 0 0 ) . The cages were m o n i t o r e d for
mating pairs at 10 rain intervals with a 40W red
light. Since moths remain in copula for about 1
- 2 h. it is unlikely we o v e r l o o k e d any mating
episodes. Mating pairs were transferred to a
small plastic container (4 cm depth • 9 cm
dia.), and termination of copulation was re-
corded.
The effect of s p e r m a t o p h o r e size on female
remating rate was evaluated by allowing
females to mate with males which were esti-
mated to have transferred large or small sper-
matophores. F e m a l e s of each type (solitary and
gregarious) were r a n d o m l y assigned to each of
2 treatments: mating with 4- to 6-day old virgin
males or males that had m a t e d once 1 o r 2 days
before (hereafter defined as virgin and m a t e d
males, respectively). F o r solitary-type males.
s p e r m a t o p h o r e Size (Ss) can be estimated by a
regression equation (He and Tsubaki. in press),
mating (days)) for virgin males, and by a n o t h e r
regression equation, Ss - 0.279 + 0.166T (T in-
dicates days since first mating) for m a t e d males.
F o r gregarious-type males, we used Sc = 0.580
+ 0.045A and Sc -= 0.489 + 0.079T for virgin
and m a t e d males, respectively. F r o m these
equations we o b t a i n e d 4 estimated size ranges
of s p e r m a t o p h o r e s p r o d u c e d by the virgin and
m a t e d males of the solitary and gregarious
types (Table 1). All females were 3 - t o 6-day
old in the first mating. It was known that this
was the age at which females were most likely
to mate for the first time.
M a t e d females were released every day into a
mating cage with the same n u m b e r of virgin
males, and remating was observed. T h r e e - d a y
old virgin males were used. R e m a t e d females
were t a k e n out from the cage and dissected to
m a k e sure that they had actually received 2
spermatophores.
In every case of the mating e x p e r i m e n t s , both
types of females were allowed to mate with
thier own types of males.
Results and Discussion
Table 1 shows proportions of females that
had r e m a t e d b e f o r e the fourth day after the

Table 1. Effect of spermatophore size on female remating

Second No, of Spermatophore received Cumulative

at the first mating remating
ratio on
mating* females type* * size (rag) * * * 4th day

Gf-Gm 19 large (GL) 0.76-0.85 0.316" * * *

!8 small (GS) 0,57-0.65 0.556

Sf-Sm 17 large (SL) 0.65-0.78 0.471

23 small (SS) 0.45-0,61 0,609

Gf, Gm--gregarious-type female and male, respectively;

Sf, Sin--solitary-type female and male, respectively.
GL, GS (or SL, SS)--gregarious-type (or solitary-type) females estimated, at
the first mating to have received large and small spermatophores, respectively.
estimated in a previous paper (He and Tsubaki, in press)
This value was significantly different from the value of GS at 5% level and
that of SS at 1% level (one-tailed binomial test).

0.7"
_~O ...... ,'O SS(n=23)
0.6"
. . "o'''~" . . . - [ 3 GS(n=18)
E
w~ 0.5"
O /O .-"" O oSL(n=17)
o
0.4'
13.
o
GL(n 19)
0.3'
0.2"
~3
0.1"
0,0
Days after Previous Mating
Fig. 1. Cumulative proportions of remated females in
relation to spermatophore size received from the first
mates. GL and GS indicate gregarious-type females
which were estimated, at the first mating, to have re-
ceived large and small spermatophores, respectively,
while SL and SS are the solitary-type females estimated
to have received large and small spermatophores, respec-
tively.
first mating with males of different types and
mating experiences. G L and GS indicate
gregarious4ype females which were estimated,
at the first mating, to have received large and
small spermatophores, respectively, while SL
and SS are the solitary-type females estimated
to have received large and small sperma-
tophores, respectively. The proportion of re-
mated females among GL-mated females was
lower than that for GS-mated females. This pat-
tern was also seen between females of SL and
SS matings. The difference of cumulative
female remating ratio on the fourth day after
the first mating was significant between GL and
G S matings of gregarious-type females (bino-
mial test, p < 0.05), but not between SL and
SS matings of solitary-type females (binomial
test, p > 0.05) (Table 1). Figure 1 shows trends
in cumulative proportions of remated females.
Although the difference was not significant,
solitary-type females tended to remate in higher
proportion than gregarious-type females.
In many butterflies and moths, effects of
spermatophore size on male and female repro-
ductive success have been suggested. Although
the effect of spermatophore size on female
49
fecundity is controversial (see Oberhauser,
1989), there is little disagreement on the effect
of the spermatophore on delaying female re-
mating. Delayed female remating is beneficial
to the first male, if some proportion of eggs was
fertilized by sperm of the second male (Drum-
mond, 2984), Many studies reported that large
spermatophores delay female remating (e.g.,
Rutowski, 1980; Rutowski et al., 1981;
Oberhauser, 1989). This experiment is another
example of this phenomenon.
Traditionally, the phase variation in army-
worms has been considered to be an adaptation
for coping with highly variable environments
(Iwao, 1968). Biological characteristics such as
fast developmental rate, tolerance to unpalat-
able food sources, and tolerance to starvation
have been known in gregarious-phase larvae,
Our study suggests that there may be differ-
ences in sperm competition tactics between
adult males of the 2 types. When larvae de-
velop under crowded conditions, adult popula-
tions are also likely to be overcrowded. It is
possible that the high moth density makes
females more likely to remate. Larger sperma-
tophores in gregarious-type males may be an
adaptation to prevent female remating at high
population densities.
It is interesting that solitary-type females
were more likely to remate than gregarious-type
females. This may be due not only to the small
spermatophore of solitary-type males but also
possibly because the volume capacity of the
bursa copulatrix may be larger in solitary-type
females than in gregarious-type females.
However, our experiments have been done us-
ing males and females belonging to the same
type, and sample size was not large enough to
answer this question at this stage of study. We
need to conduct further mating experimetns, in-
cluding mating pairs of different types.
Acknowledgements - - We thank Dr. S. Yagi,
National Institute of Sericultural and Entomolo-
gical Science, Tsukuba, for offering the army-
worm stock and teaching us the artificial diet
rearing method. Thanks are also due to Prof.
Y. Itr, Nagoya University, for critical reading
of the earlier versions of the manuscript.
50
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