ARTICLE IN PRESS

Journal of Insect Physiology 50 (2004) 11631170

www.elsevier.com/locate/jinsphys

Production of polymorphic sperm by anopheline mosquitoes and

their fate within the female genital tract
Marc J. Klowden, Gail M. Chambers
Division of Entomology, University of Idaho, P.O. Box 442339, Moscow, ID 83844-2339, USA
Received 23 July 2004; received in revised form 18 October 2004; accepted 20 October 2004

Abstract
The males of two mosquito species within the Anopheles gambiae complex, An. gambiae s.s. and An. quadriannulatus, as well as
males of An. darlingi, produced sperm of signicantly varying lengths, while a sperm polymorphism was absent in Aedes aegypti and
other anophelines not suspected of belonging to species complexes. The polymorphic distribution of these sperm lengths was not
signicantly different in smaller adult males that were reared on a low larval diet. The reproductive tract of the female was more
likely to contain larger sperm, but overall sperm retention varied depending on the size of the female and the volume of the
spermatheca she contained. The presence of a sperm polymorphism may be a factor that has promoted speciation, as well as
providing an indication that females may mate multiply.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Sperm; Polymorphism; Sexual selection; Anopheles; Gambiae; Darlingi; Quadriannulatus

1. Introduction
The gametes produced by individual female insects
tend to be similar in structure, but there is often
considerable variation in the morphology of those
produced by the male. For example, in lepidopteran
males two distinct types of spermatozoa are produced.
The eupyrene sperm are the conventional type that
fertilize the eggs, while the apyrene type have no nuclei
and, although transferred to the female in large
numbers, have no involvement in fertilization (Friedlander, 1997; Friedlander and Gitay, 1972). A sperm
polymorphism has also been identied in hemipterans
(Kubo-Irie et al., 2003), coleopterans (Green, 2003),
orthopterans (Morrow and Gage, 2001), hymenopterans
(Lee and Wilkes, 1965) and dipterans (Joly and
Lachaise, 1994; Presgraves et al., 1999; Snook et al.,
1994; Ward and Hauschteck-Jungen, 1993) in which
Corresponding
author.
Tel.:
+1 208 885 7546;
+1 208 885 7760.
E-mail address: mklowden@uidaho.edu (M.J. Klowden).

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0022-1910/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2004.10.008

nucleated sperm of various sizes participate to varying

degrees in egg fertilization. In the males of the
Drosophila obscura group, two general types of sperm
are produced. Short and long sperm types are
distinguished by their overall head and tail lengths and
there is further variation in length within each of these
categories (Snook, 1997). Although both sperm types
are motile and nucleated, the longer sperm are more
likely to fertilize eggs (Snook et al., 1994).
Following copulation, female insects store sperm in a
receptacle and the sperm are parceled out to fertilize the
egg shortly before it is oviposited. There may be
differences in the size of these receptacles among the
females of a species (Miller et al., 2001; Pitnick et al.,
1999; Pitnick and Miller, 2000), and the environment of
the female reproductive tract (Keller and Reeve, 1995;
Pitnick et al., 2003) as well as the programming of the
female nervous system (Arthur et al., 1998) may select
for the success of certain sperm types. The correspondence between rapidly evolving sperm types in the male
and sperm receptacles in the female has been proposed
to be a consequence of postcopulatory sexual selection

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M.J. Klowden, G.M. Chambers / Journal of Insect Physiology 50 (2004) 11631170

(Eberhard, 1996; Pitnick et al., 2003), and may be one of

the mechanisms of reproductive isolation that ultimately
leads to speciation (Miller et al., 2001).
In female mosquitoes, the switchover to the mated
state was thought to be under the control of male
accessory gland substances (Craig, 1967), but it was
recently determined that in several medically important
anopheline mosquitoes, these substances are not responsible (Klowden, 2001). Consequently, we focused
on another component of the ejaculate, the sperm, as a
potential trigger for the switchover to the mated state
in these anophelines. While doing so, we discovered a
previously unidentied polymorphism in their sperm.
Here we describe the rst evidence of a sperm
polymorphism in mosquitoes, within two members of
the Anopheles gambiae species complex and in An.
darlingi, and report the differential movement of these
sperm through the female genital tract of An. gambiae.

2. Materials and methods

2.1. Mosquito stocks and rearing
We used laboratory-reared Aedes aegypti (Linnaeus)
UGALS strain, An. albimanus Wiedemann, An. freeborni Aitken, An. gambiae Giles sensu stricto and An.
quadriannulatus Theobald mosquitoes. Field-collected
eggs of An. darlingi Root from Amazonian Brazil were
hatched in the laboratory and the resulting adult
mosquitoes were also examined. Larvae were reared in
shallow pans in cohorts of 200 in 450 ml water and were
fed increasing amounts of diet daily according to
species. Ae. aegypti larvae were fed a diet of nely
ground rat chow, brewers yeast and lactalbumin
hydrolysate (1:1:1 by weight). Anopheles larvae were
fed nely ground Tetramins tropical sh food. To
determine if there were nutritional effects on sperm
distribution, we also reared mosquitoes on a low larval
diet (Takken et al., 1998) that resulted in smaller adults
with reduced nutritional reserves. Adults were provided
with 10% sucrose solution continuously available from
cotton wicks. All stages were maintained at 27 1C, 70%
RH and a 14:10 h L:D cycle with 1 h dawn and dusk.
2.2. Sperm measurements
The reproductive organs of 2-day-old males were
dissected in phosphate-buffered saline (PBS). Sperm
were dispersed from one seminal vesicle of Ae. aegypti
males. Because sperm are not stored within the seminal
vesicles of Anopheles males, one whole testis was
transferred to 7 ml of fresh PBS, the cysts torn open,
and the sperm bundle teased out and gently dispersed.
Glycerol (1 ml, 1% aqueous) was added to prevent the
saline from crystallizing as it dried, and an 18 mm cover

glass was then applied. All distinct, intact sperm were

located at 100  using dark eld illumination and then
imaged at 400  using phase contrast illumination. A
microscope-mounted video camera was used to transmit
the images to a large monitor, where images of the
sperm were measured using a planar tool. Frequency
distributions of sperm lengths in 50 mm increments were
determined using SigmaStat 2000 software (Jandel
Corporation). Statistical differences between experimental groups were determined by transforming percentages
to their arcsine values and testing for their equality
(Sokal and Rohlf, 1969). The sperm from 3 to 5 males
for each species were measured.
To measure the sperm within females, spermathecae
were rst removed, isolated and cleaned in a drop of
PBS on a glass slide, and carefully transferred to 7 ml of
fresh PBS. They were gently cracked open and their
contents expressed and teased apart from using ne
needles. The spermathecal capsule was broken apart to
separate sperm that adhered to its inner surface. Before
applying the cover glass, the preparation was checked at
100  using dark eld illumination, and the spermatheca and its contents were teased apart more if necessary.
Glycerol was added, a coverslip applied, and the sperm
were measured as described above. The diameters of the
spermathecae were also measured and their dimensions
converted to volumes, with the assumption that their
round shape approximated that of a sphere.
The sperm within the female reproductive tract were
measured in An. gambiae females that were dissected
within 15 min after exposure to males. Newly mated
females were identied by the presence of a mating plug
in the common oviduct and the absence of sperm in the
spermatheca. The common oviduct was immediately
dissected and the portion just anterior to the mating
plug was opened in 7 ml PBS to disperse the newly
deposited sperm mass. Glycerol was added and a cover
glass applied as described above. Slides were examined
at 100  under dark eld illumination and all distinct,
intact sperm were individually imaged at 400  as
described previously.
Sperm were examined for the presence of DNA by
uorescence staining. Sperm from 2-day-old unmated
males were stained with 2.0 mM Hoechst 33342 stain in
PBS and examined with an epiuorescence microscope
for the presence of the nuclear stain. Only the presence
or absence of DNA was observed; the relative quantity
of DNA was not measured.

3. Results
3.1. Sperm length polymorphisms
The sperm of Ae. aegypti, An. albimanus, and An.
freeborni were fairly uniform in length. More than 65%

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of the sperm from Ae. aegypti males were in the

250300 mm category, with very slight variation (Fig. 1).
Similarly, of the sperm from An. albimanus males, more
than 85% fell within the 50100 mm category. In An.
freeborni males, more than 65% of the sperm were in the
50100 mm range.
The two members of the An. gambiae species complex,
An. gambiae s.s. and An. quadriannulatus, showed
considerable variation in sperm lengths measured from
males. In An. gambiae, sperm lengths varied from 26 mm
to over 500 mm; in An. quadriannulatus, they ranged in
length from 40 to over 450 mm. The distribution was
slightly different in the two species, with about 57% of
An. gambiae falling within 250350 mm, and 44% of An.
quadriannulatus in the 300400 mm range. When An.
gambiae were reared on a low larval diet so as to reduce
the size of the emerging adults, the sperm distribution of

1165

the smaller males did not differ signicantly from that of

larger males (Fig. 2).
Adult An. darlingi that emerged from eld-collected
eggs also showed a variation in sperm length. About
55% of the sperm were 200250 mm in length, but the
three smaller categories also each contained about
610% of the total sperm (Fig. 1).
3.2. Selective transfer and retention of sperm by females
Compared to An. albimanus and An. freeborni, species
that produced smaller, non-polymorphic sperm, the
larger polymorphic sperm from An. gambiae, An.
quadriannulatus, and An. darlingi males were associated
with signicantly larger spermathecae in females
(Fig. 3). The smaller spermatheca of An. freeborni
contrasted with its larger overall body size. An. gambiae

100

Aedes aegypti

80
60
40
20
0
100
80

Anopheles albimanus

60
40

Percent of sperm in length category

20
0
100

Anopheles freeborni

80
60
40
20
0
100

Anopheles quadriannulatus

80
60
40
20
0
100

Anopheles darlingi

80
60
40
20
0
100

Anopheles gambiae

80
60
40
20
0

50

100

150

200

250

300

350

400

450

Upper bound of length category (m)

Fig. 1. Distribution of sperm lengths in the testes and seminal vesicles of six mosquito species. Sperm lengths were divided into 50 mm categories.

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M.J. Klowden, G.M. Chambers / Journal of Insect Physiology 50 (2004) 11631170

1166

Percent of sperm in length category

100
Large gambiae
Small gambiae

80

60

40

20

0
50

100

150

200

250

300

350

400

450

Upper bound of length category (m)

Fig. 2. Distribution of sperm lengths in large and small An. gambiae males.

16
14

Sperm length
Spermatheca volume

Spermatheca volume X 10 (m )

18

400

Sperm length (m)

300

12
10
200

8
6
100

4
2
0

An. gambiae

An. gambiae An . quadriannulatus An. darlingi

(Low diet)

An.albimanus

An. freeborni

Fig. 3. Mean lengths of sperm from the males of ve mosquito species and smaller (low larval diet) An. gambiae (black bars) and the spermathecal
volumes of corresponding females (gray bars). Vertical lines represent one standard error.

reared on the high larval diet had signicantly larger

spermathecae than those smaller adults reared on a low
larval diet (Fig. 3).
When males reared on a high diet were allowed to
mate with either large or small females, the retention of
polymorphic sperm differed depending on the size of the
female. Smaller sperm of less than 50150 mm were
signicantly less likely to be present in the spermathecae
of either large or small females (Fig. 4). The spermathecae of small females contained a signicantly greater
proportion of 200250 and 300350 mm sperm, while
those of larger females contained almost twice the
proportion of 250300 mm sperm compared to male
production (Fig. 4).
Fig. 5 shows a box plot of sperm lengths in An.
gambiae male testes, within the oviduct of females, and
within the female spermathecae. The distributions of
sperm lengths in the female oviduct soon after mating
and within the spermatheca afterward were considerably
reduced compared to those in the male testis, indicating

that the selection for long sperm was not simply based
on the acceptance by the spermatheca but on the sperm
that were transferred during insemination.
3.3. Fluorescence staining for DNA
Sperm from An. gambiae males that were polymorphic for tail lengths were identied using phase
contrast illumination and then examined for uorescence. All sperm examined, ranging from very short to
very long tail lengths, showed uorescence corresponding to the head that indicated DNA was present.

4. Discussion
The two members of the An. gambiae complex that we
examined, An. gambiae s.s. and An. quadriannulatus,
both produced polymorphic sperm with a wide range of
tail lengths. Individual males of both species formed a

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M.J. Klowden, G.M. Chambers / Journal of Insect Physiology 50 (2004) 11631170

Percent of sperm in length category

60

% in length category:
large females
% in length category:
small females
% in length category:
male production

50

1167

*
*

40

30

20

10

50

**

**

100

150

200

250

300

350

400

450

Upper bound of length category (m)

Fig. 4. The distribution of sperm lengths in male An. gambiae (gray bars) and in large (black bars) and small (white bars) females. Asterisks indicate
signicant differences (Po0.05) in the proportions of sperm within a size category compared to the proportion produced by males.

Sperm length (m)

400

300

200

100

0
Testis

Oviduct

Spermatheca

Fig. 5. Box plots of the sperm lengths from the testes of male An.
gambiae and from the oviducts and spermatheca of females. The line
within each box indicates the median and boundaries of the boxes
indicate the 25th and 75th percentiles. Vertical lines indicate the 10th
and 90th percentiles.

variety of sperm in the range of 26503 mm, in contrast

to Ae. aegypti, An. albimanus and An. freeborni that
produced sperm of lengths within a much narrower
range (Fig. 1). In An. gambiae at least, all of the sperm
we examined were nucleated, in contrast to the sperm
dimorphism found in lepidopterans in which the larger
eupyrene sperm are produced along with the smaller
anucleated apyrene sperm. We considered the likelihood
that the sperm lengths actually represented a range of
maturational stages that were examined before they had
fully matured, but all had distinct heads and tails,
evidence that they had completed spermiogenesis. The
possibility that the polymorphism represented sperm
whose tails had been truncated during preparation was
also dismissed, because the few broken sperm we

observed were readily identiable by the broad ends of

their tails, compared to the more tapered ends of intact
sperm. Also, the presence of mature polymorphic sperm
in the reproductive tracts of females indicated that the
polymorphism observed in sperm from the testes was
indeed real. Whether the sperm polymorphism in An.
gambiae might be a laboratory artifact resulting from
long-term colonization was also considered, but the
polymorphism was also present in newly collected An.
darlingi that had never been colonized. The sperm
polymorphism did not appear to be affected by adult
size or larval diet, as the pattern of polymorphic sperm
production did not differ in adult males reared on either
standard or low diets (Fig. 2).
The most intriguing implications of these data relate
to the possible roles of the polymorphic sperm and
reasons for the evolution and maintenance of sperm
types that may not all have the same probability of
participating in fertilization. A sperm polymorphism has
been observed in many other insects, with sperm
competition considered as the major force driving its
evolution (Parker, 1970). In sperm competition, the
ejaculates of two or more males compete to fertilize the
eggs of a female, with the reproductive tract of the
female performing a post-copulatory selection for those
sperm that will be used to fertilize the eggs. This
selection tends to favor larger sizes when the sperm
competition is more intense, as demonstrated both in
insects and nematodes (LaMunyon and Ward, 2002;
Miller and Pitnick, 2002). Although the larger sperm
tend to be more metabolically expensive for the male to
manufacture (Pitnick et al., 1995), they may be, as
described by Miller and Pitnick (2002), the cellular
equivalents of the peacocks tail, as the female

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M.J. Klowden, G.M. Chambers / Journal of Insect Physiology 50 (2004) 11631170

reproductive tract selects for larger sperm to incorporate

into her eggs (Bressac and Hauschteck-Jungen, 1996;
Snook and Karr, 1998). Of course, depending on the
mating systems, sperm size also has been negatively
correlated with fertilization success (Gage and Morrow,
2003). Species isolation, followed by a divergence in
sperm and female reproductive tract morphology and
the components that comprise male accessory gland
secretions (Pitnick et al., 1991, 1999; Pitnick et al.,
1997), could provide the raw materials upon which postcopulatory sexual selection might act (Parker, 1970). In
these cases, there may be some circumstances where the
size of sperm may affect the outcome of sperm
precedence when the sperm of several males are present
in the spermatheca (Price, 1997).
The morphology of the female reproductive tract is
particularly important with regard to sperm competition
resulting from multiple mating (Bangham et al., 2003;
Joly et al., 1991). Miller and Pitnick (2002) observed
that the success of male Drosophila in fertilizing eggs
was related to the interaction between polymorphic
sperm and the morphology of the female sperm storage
organ. We found that the size of the spermathecae in An.
gambiae varied when females were reared on a low diet
and this variation may have been responsible for the
selective retention of some sperm sizes by small and
large females (Fig. 4). Spermathecal volume was
correlated with the overall size of the female; low diet
An. gambiae females had signicantly smaller spermathecae than those larger females that were reared on
the high diet. However, even the low diet An. gambiae
contained signicantly larger spermathecae than did An.
freeborni, which had a much larger body size, and An.
albimanus, both of which had males that did not
produce polymorphic sperm.
In An. gambiae, the size distribution of sperm differed
between what was produced by the male and what was
found in the genital tract and spermatheca of the female.
The smaller spermathecae of low diet females tended to
retain a signicantly larger percentage of sperm in the
length categories of 250 and 350 mm, whereas larger high
diet females tended to retain signicantly more sperm in
the 300 mm length category relative to their proportional
production by the male (Fig. 4). Smaller sperm in the
size categories under 150 mm were signicantly less
represented in the spermathecae of females of both
sizes. The selection for larger sperm appeared to have
occurred before the sperm entered the spermatheca, as
the range of sperm sizes was already reduced by the time
they were present in the common oviduct (Fig. 5). We
do not know the extent to which the smaller sperm in the
spermatheca are actually involved in fertilization.
One of the driving forces for sperm polymorphism is a
post-copulatory sperm competition among the sperm
that are present from more than one male (Clark, 2002),
and if this mechanism is operating in An. gambiae, it

suggests that multiple mating by females might be a

relatively frequent phenomenon. A relationship between
sperm polymorphism and female mating frequency has
already been suggested in Drosophila (Bressac et al.,
1991) and in lepidopterans, where the smaller sperm
may serve as cheap ller that delay the emptying of
the spermathecae that serves as the trigger for remating
(Cook and Wedell, 1999; Gage, 1994). Although it has
long been assumed that female mosquitoes mate only
once because peptides in male accessory gland substances assure monogamy (Craig, 1967), we have
recently shown that the peptides produced in the male
accessory glands of several anophelines lack the ability
to prevent a subsequent mating by the female (Klowden,
2001) but mating does affect female fecundity (Klowden
and Russell, 2004). Whatever evidence exists for
polyandry in eld populations of mosquitoes has indeed
come from anopheline populations only (Tripet et al.,
2003; Yuval and Fritz, 1994), although the extent of this
multiple mating by these natural populations is relatively low.
The rapid evolutionarily divergence of male reproductive products and the coevolution of female reproductive tract morphology may provide the basis for
reproductive isolation and serve as an engine of
speciation (Pitnick et al., 2003). Of the three Anopheles
species that we found to have a sperm polymorphism,
two are known to be part of a species complex, and the
status of the third, An. darlingi, has yet to be adequately
resolved (Conn, 1998; Harbach et al., 1993; Lounibos
and Conn, 2000; Manguin et al., 1999). We speculate
that the sperm polymorphisms we observed in these
anophelines may also be one characteristic that marks
the existence of a species complex. This recognition is
particularly important for insect vectors that may be
morphologically identical but vary behaviorally or
physiologically and thus affect parasite transmission.
How might differences in sperm morphology be
translated to trends toward speciation? Sperm tails
persist within the yolk of Drosophila eggs after fertilization occurs (Bressac et al., 1994; Karr, 1991; Karr and
Pitnick, 1996) and even remain within the midguts of
developing larvae (Pitnick and Karr, 1998). If their tails
vary in size, they may also provide different paternally
derived components to the developing oocytes. The
physiological effects of sperm on the reproductive
behavior of the female Drosophila receiving them lie in
part with the ability of the sex peptide to bind to sites on
the sperm tail and be released once it resides in the
female (Liu and Kubli, 2003). If a similar mechanism
also occurs in An. gambiae, longer sperm may provide
more opportunities for the binding of male products for
transport into the egg than can shorter sperm, and as the
sperm evolve to greater lengths, these can consistently
provide raw materials that will be reected in the
differences in offspring.

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In addition to a sperm polymorphism, the differences

in reproductive structures between some anopheline and
aedine mosquitoes are noteworthy. Many anopheline
vectors have only one spermatheca, no bursa copulatrix,
little or no storage of sperm in the seminal vesicles, and
the production of a mating plug in the female from male
accessory gland secretions that appears to mechanically
prevent multiple mating, rather than peptides that
ensure monogamy physiologically (Craig, 1967; Giglioli,
1963; Giglioli and Mason, 1966; Klowden, 2001). A
number of other physiological differences between the
two mosquito genera have also been observed (Briegel,
2003; Briegel and Horler, 1993; Timmermann and
Briegel, 1999). Thus, although much of what we know
about mosquito biology is based on an Aedes model,
anophelines, the most important vectors of parasites to
humans, appear to deviate from that model in several
ways. These considerations illustrate the inaccuracies of
generalizing about mosquito reproduction without
specifying the species in question, and will be particularly important if it ever becomes possible to introduce
genes for refractoriness to parasite infection using male
mosquitoes as the vehicle for gene dissemination into
eld populations.

Acknowledgments
We are grateful to Troy Ott and Kurt Gustin for their
assistance with the epiuorescence microscope and
advice about technique. The anopheline colonies used
in this research were graciously provided by Professors
C. Curtis, W. Takken, and J. Conn. This research was
supported by grant IBN-0210251 to MJK from the
National Science Foundation.

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