Ae. eLaoprcrus REPRoDUcTIoN
JUNE1993
r-69
MATING AND NUTRITIONAL STATE AFFECT THE
REPRODUCTIONOF AEDES ALBOPICTUS MOSQUITOES
MARC J. KLOWDEN
Diuision of Entomology, [Jniuersity of ldaho, Moscow,ID 83844
ABSTRACT. Mated femaleA edesalbopictusmosquitoesthat were maintained after emergencewithout
carbohydrate were more likely to develop eggs after a small blood meal than were unmated females
on the same diet. The effect was-dueto male accessorygland substancestransferred to the
-"i"tui""a
female during mating. Neither the endogenousreserves of protein-and lipid. nor the number of eggs
developedpei volum6 of ingested blood differed between mated and unmated females,-suggesting-that
ttt" "titi*ttio" of existing ti."*"* was altered by mating. Methoprene administered to both mated and
""-ut"a females that irigested small blood meals reduied the likelihood that egg development would
occur. Small volumes of b"loodwere more Iikely to trigger oogenesisin both mated and unmated females
if their abdomenswere additionallv distended.
INTRODUCTION
The mosquitoesAedes aegypti (Linn.) and
Aedesalbopictus(Skuse)are related speciesthat
occupy similar ecologicalniches. The distribution of both specieshas increasedas a result of
their ability to colonizecontainersin and around
human habitats. When their distributions have
overlapped,one of the 2 specieshas generally
been supplanted (Gilotra et al. 1967,Mogi et al.
1988,Hobbs et al. 1991),but the ecologicalcircumstancesunder which one speciesmight be
replacedby another are difficult to predict. The
competitive interactions between the larvae of
these specieshave most often been used to explain their distribution patterns (Macdonald
1956,Moore and Fisher 1969,Black et al. 1989).
When maintained on carbohydrate-restricted
diets, Aedesalbopiretusis more likely to produce
eggs after ingesting small blood meals than is
Aedes aegypti (Klowden and Chambers 1992).
Although some of this difference may be due to
the longer Iarval period of Ae. albopirtus thaL
allows it to accumulate greater lipid and glycogen reserves,it appearsthat the adult reproductive metabolism of the two speciesis different,
a difference that may partially explain the outcome of competitive interactions. In this study,
the reproductive metabolism of Ae. albopicttt's
was further explored, with an examination of
the relationship between nutritional state and
the male accessorygland substancesthat are
contributedduring mating.
MATERIALS
AND METHODS
period, and starved adults were maintained at
80VoRH with accessto only water from cotton
wicks. Sugar-fedadults were fed a 10% sucrose
solution. Approximately l0% of the stock population developeda first batch of eggs autogenously, but only when the femaleswere provided
with sucrose after emergence.Autogeny was
never observedin the experimental groups that
did not ingest carbohydrate.
Accessoryreproductive glands were removed
from 4-day-oldunmated,sucrose-fedmalesand
implanted into the abdomens of unmated femalesthrough a slit in the arthrodial membrane.
The wounds were sealedwith molten paraffin.
In controls, the arthrodial membrane was
opened and immediately sealed.Adult females
were given measuredamounts of heparinizedrat
blood administered as enemas(Briegel and Lea
1975). They were dissectedand examined for
eggs2 days later.
Nutritional reservesof carbohydrateand lipid
in individual mosquitoes were assessedby the
methods of Van Handel (1985a, 1985b) performed as describedpreviously (Klowden and
Chambers 1992). Total protein in mated and
unmated females was determined by first sonicating individual mosquitoes in 0.01 N NaOH,
centrifuging to pellet insoluble portions, and
assaying an aliquot of the supernatant by the
method of Bradford (1976)basedon a standard
curve of bovine serum albumin.
Statistical differences between the responses
of experimental groups were determined by
transforming percentagesto their arcsine values
and testing for their equality (Sokal and Rohlf
1969).Regressionlines were comparedby the
methodof Gomezand Gomez(1984).
The Ae. albopictuscolony was obtained from
individualsoriginally colonizedin Lake Charles,
Louisiana. Larvae were reared at 27"C and fei
RESULTS
daily until pupation with increasing amounts of
More than 90% of the mated Ae. albopictus
powdered rat chow-lactalbumin hydrolysatebrewersyeast (1:1:1by weight).Both larvaeand females that were maintained on water for 4
adults were raised under a 14:10 (L:D) photo- days after emergenceand then fed 1 1tl or more
170
Jounull oF THEAunRrclx Moseurro Cor.rrnor,AssocrlrroN
ofblood developedeggs.Oogenesis
was initiated
in about207oof the mated population even when
as little as 0.1 pl of blood was ingested(Fig. 1).
However, unmated females had a much higher
threshold for oogenesis,requiring 0.25pl or more
of blood before eggswere produced. Increasing
volumes of blood up to 1 pl initiated oogenesis
in an increasingproportion of the unmated population, but significantly fewer developed eggs
compared with mated females given the same
volume of blood (Fig. 1). Although there appeared to be significant differences in the responseof mated and unmated females to blood
ingestion, the relationship between the number
of eggs produced and the volume of blood ingesteddid not differ significantly betweenmated
and unmated females (Fig. 2).
To determine whether the difference in their
responseto blood ingestion may have resulted
from differences in their nutritional reserves.
mated and unmated females were analyzed for
total glycogen,Iipid and protein. Glycogencontent was significantly greater in unmated females, but the low Ievels found in both mated
and unmated females were not biologically
meaningful. In contrast, there were no significant differences between the much larger lipid
and protein reserves in mated and unmated
females(Table 1).
Becauseit appearedthat mating affected egg
development,single accessoryglands from male
Ae. albopictuswere implanted into unmated Ae.
albopictusfemales,which were then given 0.5 prl
of blood by enema.As shown in Table 2, the
implantation of the glands significantly increasedthe ability of the unmated females to
(/j.^^
rb
rh
rtl
IUU
; 8 0
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F
f--_UNMATED
SSIMATED
VoL. 9, No. 2
720
100
frl
*l
80
rr'l
60
lr'l
(h
40
rh
r r'l
20
.5
jl
0
BLOODMEAL SIZE (/rl)
Fig. 2. Effect of increasingblood meal size on the
number of eggs developing per female in mated and
unmated Aedes albopictus. The regression lines for
mated (Y : 17.6 + 22.5X; 12: 0.98) and unmated
(Y : 25.6 + 16.9X; r' : 0.92) femalesdid not differ
significantly(s:2.04; P > 0.05).Vertical bars represent standard errors.
Table 1. Whole body glycogen,lipid, and protein of
mated and unrnatedAedesalbopictusfemales
maintained on water for 4 days following emergence.
Micrograms/female+ SE
Lipid
Protein
+
+
Mated
5.5 0.7'* 166.7 5.7" 254.t + 2.7^
Unmated 8.5 + 1.0b 174.4+ 6.2" 255.9+ 2.4"
Treatment
Glycogen
* Values followed by the same letter within a column ate not significantly different (P > 0.05); n : 60
for all treatments.
3 6 0
rTl
rrt
< T U
F
zl.J)
Table 2. Effect of whole male accessorygland (MAG)
implants ftom Aedesahopictus or Ae. aegypti into
unmated Ae. albopictusfemales.Following
implantation, the hosts were given 0.5 pl ofblood.
zu
U
H o
0.10 0.2s 0.50 1.00 2.00 3.00 4.00
BLOODMEAL SIZE (#,1)
Fig. 1. Effect of increasing blood meal size on the
percentage of mated and unmated Aedes albopictus
that developedeggs.The percentagesfor mated and
unmated females differ significantly (P < 0.05) with
blood volumes of 1 pl and below. Vertical bars represent standard errors.
Percentage
with eggs
Treatment
(+sE)
4l
22.7 + 6.3
Sham-operatedcontrols
MAG from Ae. albopicttts 47.6 + 10.9* 21,
OK
MAG from Ae. aegypti
13.3 a 8.8
* Indicates a significant differencefrom controls (P
< 0.05).
An.,qr,eoprcrus REPRoDUcrloN
JUNE 1993
develop eggs.However, unmated Ae. albopictus
implanted with a male accessorygland from Ae.
aegyptiwere not affected, and their responseto
blood did not differ significantly from shamoperatedcontrols.
Abdominal distention provides nervous signals that affect oogenesisdifferently in mated
and unmated Ae. aegypti (Klowden and Chambers 1991).Mated and unmated Ae. albopictus
were given either their threshold blood volumes
for oogenesis(0.1 pl for mated and 0.25 pl for
unmated). or an additional volume of saline
along with the blood to provide a total meal
volume of 1 pl. The percentagewith egg development was increased in both mated and unmated femalesby the additional abdominal distention from the extra saline in the meal (Table
3).
To determine whether the juvenile hormone
analog methoprene affected egg development,
either 1.0 or 0.1 pg of methoprene was applied
to the abdomens of mated and unmated mosquitoes shortly after they were given 1 pl of
blood. As shown in Fig. 3, topical application of
methoprene reduced the proportion of both
mated and unmated Ae. ahopictus that developed eggsfrom the blood.
rh 100
L--_]UNMATED
r h
f r'l
NSluerso
80
rT{
F
=
60
rYl
rla
40
li
z
frl
U
20
rYl
DISCUSSION
Components of the male accessoryreproductive gland affect the reproductive metabolism of
starvedAe. albopictusfemales,allowing them to
redirect resourcesand develop eggsthat would
otherwisenot be produced(Fig' 1). Theseaccessory gland components do not appear to act by
affecting the pool of metabolic reserves,because
although glycogen levels were actually greater
in unmated females, perhaps because males
causedthe mated femalesto expendmore energy
for flight, protein and lipid levels were not significantly different (Table 1). Also, the numbers
of eggsdevelopedper unit of blood ingested did
not differ between mated and unmated females
(Fig. 2). However, mating or accessorygland
implantation increasedthe probability that eggs
0
CONTROLS0.1
1.0
METHOPRENEDOSE (Pg)
Fig. 3. Effectsof topicallyappliedmethopreneon
that were
females
matedandunmatedAedesalbopictus
fed 1 pl of blood.Controlsreceivedthe sameamount
of bloodbut weretreatedwith acetoneonly. Vertical
barsrepresentstandarderrors.
would develop from a small blood meal (Fig. 1,
Table 2). Therefore, a mechanism of male'
induced oogenesis similar to that previously
described for Ae. aegypti (Klowden and Chambers 1991) appears to also be present in Ae.
albopictus.
It is generally believedthat male-derivedsubstancesin insects are broken down and directly
utilized or incorporated into the eggs of the
female (Young and Downe 1987,Boggs 1990).
However, the present data suggestedthat in
mosquitoes, male accessoryglands specifically
affected the reproductive metabolism of the female and directed the manner in which existing
reserveswere utilized. Glands ftom Ae. aegyptl,
which were unlikely to differ significantly from
those of Ae. albopictusin the total raw materials
they contained, did not stimulate this maleinduced oogenesisin Ae. albopictus (Table 2),
although glands of Ae. albopictu.sate active in
Table 3. Effect of abdominal distention on oogenesis in mated and unmated A edes albopictus receiving
equivalent volumes of blood.
Percentage with eggs (tSE)
Treatment
Mated females
Unmated females
Blood alone*
19.7r 3.5
5.1+ 2.5
n
Blood and
saline*
61
55
46.4x.9.4
33.3I 8.2
n
28
Significance
P < 0.05
P < 0.05
* Mated females received 0.1 pl of blood or 0.1 rrl of blood plus 0.9 pl of saline. Unmated females received
0.25 palof blood or 0.25 pl of blood plus 0.75 pl of saline
r72
JounNer,oF rHE Arr,rnRrceN
Moseurro ConrRor,AssocrArron
VoL. 9, No. 2
Ae. aegypti (Klowden and Chambers 1991).A pictus colony. This research was supported in
similar result was obtained by Leahy and Craig part by grant Al-24453 from the National Insti(1965)who, in their study of male-inducedovi- tutes of Health and contribution number 92760
position, found an increasein eggdepositionin from the Idaho Agricultural Experiment Staunmated Ae. albopictusfemales implanted with
tion.
Ae. albopictusmale accessorygland, but not with
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sonal shifts in relative abundanceof Aedesahopicquality of host blood is suboptimal.
ACKNOWLEDGMENTS
I thank G. M. Chambers for her technical
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