Malaria transmission,
sex ratio and
erythrocytes with
two gametocytes
Roger Jovani
Transmission of haemospororin parasites (phylum Apicomplexa) needs the
fertilization of at least one female by one male gamete within the bloodmeal of
a suitable vector. Male and female gamete precursors (gametocytes) in
Plasmodium and Haemoproteus parasites are normally alone inside the
erythrocytes of the vertebrate host, but they also occur in male–female pairs in
single erythrocytes. These paired gametocytes could enhance transmission
success by facilitating the encounter between the female and male gametes
when inside the midgut of the vector. Further study of these particular
infections could provide new insights into the biology of and control strategies
for haemospororin parasites.
Roger Jovani
Dept Applied Biology,
Estación Biológica de
Doñana, Consejo
Superior de
Investigaciones
Científicas,
Avda. Ma Luisa s/n,
41013 Sevilla, Spain.
e-mail:
jovani@ebd.csic.es
With the bite of an infected blood-feeding dipteran
vector, sporozoites of haemospororin parasites
(phylum Apicomplexa; Plasmodium, Haemoproteus
and Leucocytozoon) travel to and infect the tissue
cells of the vertebrate host, and mature to an
asexual schizont stage. Merozoites then emerge
from the schizont and invade erythrocytes,
producing (sexual) gametocytes or continuing
schizogony until new merozoites are determined to
be gametocytes. For Plasmodium falciparum, all
merozoites from the same schizont are either all
sexual or all asexual [1], and gametocytes are either
all male or all female [2].
Transmission begins when a suitable vector bites an
infected host. Mature male and female gametocytes
emerge from red blood cells of the bloodmeal in the
midgut of the vector, producing up to eight mobile male
gametes (exflagellation) or differentiating into a single,
static, female gamete (rounding up). Fertilization
occurs when a male gamete finds and fuses with a
female gamete. After fertilization, the zygote
differentiates into an invasive ookinete that will
penetrate the midgut epithelium of the vector. There,
the ookinete becomes an oocyst, which will finally
produce thousands of sporozoites, infecting a new host
on a subsequent bite by the vector.
Although haemospororin gametocytes are normally
alone inside erythrocytes, double-gametocyte infections
(DGIs) occur in Plasmodium and Haemoproteus
parasites [3,4]. However, the importance of this
phenomenon for the biology and control of malaria has
not been considered. The DGI hypothesis states that the
encounter time between male and female gametes of a
male–female DGI in the midgut of the dipteran vector is
less than the time needed for those gametes emerging
from singly infected erythrocytes; therefore, in
scenarios of constrained transmission by single
gametocytes, male–female DGIs would have a key role.
In Plasmodium spp., the low density of gametocytes [5]
and the low proportion of gametocytes reaching the
ookinete stage [6] indicate that fertilization is a major
bottleneck on transmission. Thus, a male–female DGI
would enhance transmission success.
Current knowledge about DGIs
The ‘DGI hypothesis’ has two prerequisites:
(1) male–female DGIs must occur in natural infections;
and (2) male–female DGIs must be viable. DGIs have
never been reported for infections of Leucocytozoon spp.,
although studies actively searching for DGIs in these
species are needed. In natural infections of
Haemoproteus spp., mature male–female DGIs have
been reported for birds and tortoises [4,7]. DGIs are also
found in cultures of P. falciparum and other
Plasmodium spp. [3], but have not been reported in
natural Plasmodium infections. However, the static
environment into which merozoites are normally
released in vitro could not explain the lack of information
about DGIs in natural infections of some Plasmodium
spp. This is because, in in vivo infections of P. falciparum
and other Plasmodium spp., microvascular obstruction
resulting from cytoadherence (binding of infected
erythrocytes on the endothelium of capillaries and
venules) and rosetting (adherence of late-stage asexual
infected erythrocytes to uninfected red blood cells; Fig. 1)
also creates an environment with little or no blood flow
when merozoites emerge from infected erythrocytes [8].
Why, therefore, are DGIs not reported in natural
Plasmodium infections? First, gametocytaemia is much
higher in natural infections of Haemoproteus and in
Plasmodium cultures than in natural infections of
Plasmodium spp. Second, DGIs are rarely considered in
publications. For instance, in Haemoproteus columbae,
DGIs have been reported in some studies [7,9] but,
when it does occur, it is treated as an anecdotal
phenomenon [7] or is not discussed in the text even
when it does appear in the figures [9]. Alternatively,
DGIs might really not occur in natural infections of
Plasmodium spp. However, multiple asexual infections
inside single erythrocytes have been seen in many in
vivo infections of Plasmodium spp. (e.g. P. falciparum
and Plasmodium vivax [10]), and they are also produced
by two merozoites entering a given erythrocyte.
However, the important question is not whether DGIs
could occur in natural infections, but whether male–
female DGIs could occur. Two non-exclusive models
could explain how male–female DGIs could be
produced in P. falciparum (Fig. 1).
Even less is known about the viability of DGIs in vivo
than about their occurrence. However, DGIs have been
found to reach maturity in cultures of P. falciparum and
other Plasmodium spp., and in natural Haemoproteus
infections [3,7]. Moreover, some mature (stage V)
(a)
(b)
TRENDS in Parasitology
Fig. 1. Two hypothetical
models of how male–female
double-gametocyte
infections (DGIs) could be
produced by Plasmodium
falciparum in human
capillaries. Adhered and
non-adhered erythrocytes
to the schizont are
assumed to be susceptible
to merozoite invasion,
following the model
proposed in Ref. [25].
Schizonts coming from
singly (a) or doubly (b)
asexually infected
erythrocytes are indicated
in grey. Because of the lack
of information on ‘double
schizonts’ [24], they have
been assumed to produce
a similar number of
merozoites as ‘single
schizonts’. Schizonts
adhere to the endothelium
of the capillary and show
rosetting with uninfected
erythrocytes (red).
Merozoites committed to
be male and female
gametocytes are
represented by black and
white dots, respectively, so
male–female DGIs result
from those erythrocytes
(red) with a black and a
white dot.
female–female DGIs have been seen in P. falciparum
cultures and, on one occasion, one female gametocyte
from a female–female DGI has been seen to round up
(L. Baton and L. Ranford-Cartwright, pers. commun.).
It is particularly interesting that the technique used to
produce these results (Giemsa-stained smears from a
P. falciparum culture) was not designed specifically to
find DGIs or exflagellation/rounding up processes. Thus,
although they are anecdotal, these data suggest that
male–female DGIs could also be viable.
meeting in the bloodmeal [17] and (2) host antibodies in
the bloodmeal agglutinate male gametes, reducing
male-gamete motility [18]. In addition, each male
gametocyte produces few male gametes (around four in
P. falciparum), imposing a lower sex-ratio limit because
f the need to produce sufficient male gametes to fertilize
the female gametes [19].
However, there are some groups of blood-dwelling
apicomplexan parasites to which this theoretical
framework could not be applied because of ‘syzygy’ – male
and female gametocytes pairing before gametogenesis.
Syzygy occurs in blood-dwelling adeleorins and
piroplasms, and the available data supports the
prediction that syzygy favours a sex ratio of 0.5 [20].
Syzygy could be seen as an extreme point of DGI, at
which all gametocytes are in male–female DGIs. Thus,
the sex ratio is predicted to become less female biased as
the proportion of fertilizations coming from male–female
DGIs increases, maximizing the male–female
gametocyte ratio rather than the male–female gamete
ratio. If we do not take into account the importance of
fertility insurance [17], the optimal sex ratio (r*) could
be defined by Eqn 1,
r* = [(1 − F) ÷ 2]s + 0.5d
[Eqn 1]
and the lower sex ratio limit could be given by Eqn 2,
Low gametocyte densities
When they are present, P. falciparum gametocytes are
found in very low densities (146–485 gametocytes µl−1 [5]),
and a very low proportion reaches the ookinete stage (40–
1223-times reduction in in vitro cultures, i.e. out of
40 gametocytes, only one reaches the ookinete stage).
However, this does not seem to be an insuperable
problem for the transmission of P. falciparum, because
people with a gametocytaemia under the detection level
of three gametocytes µl−1 have been found to be infective
to mosquitoes [11–14], even at a similar proportion to
positive gametocyte carriers [12]. How are these very low
gametocyte levels infective? The number of gametocytes
in different bloodmeals from the same person shows an
aggregate distribution, some bloodmeals having a
fivefold higher density than the mean [15]. However, the
DGI hypothesis states that only one male–female DGI in
a bloodmeal is necessary for transmission, being much
more efficient than one male and one female
gametocyte in singly infected erythrocytes. Hence,
these two complementary processes could act
simultaneously, enhancing transmission success in
situations of low gametocytaemias.
Could DGI explain apicomplexan sex ratios? Current
theory about the sex ratio (the proportion of male
gametocytes) in apicomplexan parasites states that this
is shaped by two opposing forces [16]. The first is
inbreeding, which produces female-biased sex ratios.
The second is ‘fertility insurance’, which produces more
equal sex ratios under transmission-compromised
scenarios, because (1) the low number of gametocytes
leads to a low probability of a male and a female gamete
r* = [(1 ÷ (1 + c)]s + 0.5d
[Eqn 2]
where s is the proportion of fertilizations between
gametes that do not come from male–female DGIs
(most of them from singly infected erythrocytes),
d is the proportion of fertilizations from male–female
DGIs (i.e. d + s = 1), F is the inbreeding rate and c is
the mean number of gametes produced by each male
gametocyte (Fig. 2).
Therefore, the DGI hypothesis predicts that
haemospororin parasites will benefit from adjusting
their sex ratio depending on the value of d.
Accordingly, in Leucocytozoon parasites (which
probably have d = 0), the inbreeding probability has a
significant negative correlation with sex ratio [21].
If the DGI hypothesis is true, Plasmodium and
Haemoproteus parasites would be expected to have
d values that are much higher than the proportion of
male–female DGIs in the bloodstream, owing to a
higher fertilization efficiency of male–female DGIs
compared with single-gametocyte infections. This
could be especially relevant at low gametocyte
densities (because this lowers the probability of
encounters between male and female gametes coming
from single gametocytes, but does not affect
fertilization between gametes from a male–female
DGI) and when male gamete motility is impeded by
the action of antibodies [18] (because only male
gametes from singly infected erythrocytes, but not
from male–female DGIs, need to travel to encounter
the female gamete and therefore could be affected by
host agglutination antibodies).
Optimal sex ratio (r*)
Fig. 2. Optimal sex ratio
(r*) plotted against
inbreeding rate (F ) for
different proportions of
fertilizations coming from
double-gametocyte
infections (d ). This
assumes that a mean of
four gametes emerge
from each male
gametocyte.
0.5
d=1
d = 0.8
0.4
d = 0.6
d = 0.4
0.3
d = 0.2
d=0
0.2
0.1
0
0
0.2
0.4
0.6
0.8
1
Inbreeding rate (F)
TRENDS in Parasitology
Acknowledgements I
thank Lisa RanfordCartwright for a fruitful
discussion. Jordi Torres
first alerted me to the
existence of DGIs. I thank
Daniel Sol, Jordi Torres,
José Luis Tella,
David Serrano,
Dave Shutler and
Anna Perera for
continuous talks during
the gestation of this idea.
Andrea Kraljevic,
José María Fedriani and
Begoña Martinez
corrected the English.
Andrew Read and two
anonymous referees
improved the article.
The sex ratio of Haemoproteus spp. has been found
to be between 0.3 and 0.42, which did not show a
statistical relationship with the probability of
inbreeding [22]. In five populations with very low
prevalence (<20%), and so predicted to have a very
high inbreeding rate [21], the sex ratios were 0.35,
0.36, 0.40, 0.41 and 0.42, independent of inbreeding
rate [22]. Thus, supposing a relatively high
inbreeding rate of F > 0.5, Eqns 1 and 2 predict that
~40% of the fertilizations could come from male–
female DGIs (Fig. 2) if Haemoproteus lineages could
adjust their sex ratio. Therefore, the DGI hypothesis
predicts for the first time a sex ratio close to 0.4, and
independent of inbreeding rate, in situations with
moderate to high proportions of fertilization coming
from DGIs (d = ~0.4).
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