Content uploaded by Sandra Lass
Author content
All content in this area was uploaded by Sandra Lass on Nov 20, 2014
Content may be subject to copyright.
Different mechanisms of transmission of the microsporidium
Octosporea bayeri: a cocktail of solutions for the problem of
parasite permanence
D. B. VIZOSO*, S. LASS and D. EBERT
De´partement de Biologie, Unite´ d’Ecologie and Evolution, Universite´ de Fribourg, Chemin du Muse´e 10,
CH-1700 Fribourg , Switzerland
(Received 1 June 2004; revised 2 September 2004; accepted 3 September 2004)
SUMMARY
Periods of low host density impose a constraint on parasites with direct transmission, challenging their permanence in the
system. The microsporidium Octosporea bayeri faces such constraint in a metapopulation of its host, the cladoceran
Daphnia magna, where ponds frequently lose their host population due to ponds drying out in summer and freezing in
winter. We conducted experiments aimed to investigate the mechanisms of transmission of O. bayeri, and discuss how these
mechanisms could contribute to the parasite’s permanence in the system. Spores accumulate in the fat cells and the ovaries
of the host, and vary in morphology, possibly corresponding to 3 different spore types. Horizontal transmission occurred
through the release of spores from dead hosts, with the proportion of infected hosts depending on the spore dose. Further,
spores are able to persist outside the host both in dry and wet conditions. Vertical transmission occurred to both par-
thenogenetic and sexual offspring. The former were invariably infected, while the sexually produced resting eggs
(=ephippia) had a less efficient transmission. The parasite may be carried by the ephippia, and thus disperse to new ponds
together with the host. Together, these mechanisms may allow the parasite to endure periods of harsh environmental
conditions both outside and inside the host.
Key words: vertical and horizontal transmission, parasite permanence, spore survival, metapopulation, Octosporea bayeri,
Daphnia magna.
INTRODUCTION
Successful host-to-host transmis sion is critical for
the survival of parasites. Low host densities and
host extinction can produce severe bottlenecks for
parasites if transmission requires the presence of a
minimum density of susceptible hosts (e.g. if trans-
mission is density dependent, Anderson & May, 1978,
1981, 1986 ; Onstad et al. 1990). In microparasites
that infect new hosts via spores released to the en-
vironment, horizontal transmission depends on the
rate of spore contact with new hosts (Anderson &
May, 1981; Regoes et al. 2003). At high host den-
sities, the contact between susceptible hosts and
spores might be frequent enough for parasite per-
manence, even if spore survival in the environment
is short. At low densities, however, the contact
will be reduced and parasites with spores that die
before infecting a susceptible host will disappear
from the population. Frequent periods of low host
density may thus select for an increase in spore
longevity. A similar scenario has been recently
modelled for the Hantavirus-Vole system (Sauvage
et al. 2003). Their theoretical results confirm the
argument above: by adding survival outsid e the host,
the permanence and prevalence of the pathogen in
the population increased.
In microsporidia, spores released to the environ-
ment are characterized by a complex spore wall,
which has been suggested as an adaptation for sur-
vival outside the host (Bigliardi & Sacchi, 2001 ;
Maddox, 2002). The resistance to environmental
stress (often referred to as environmental persistence)
has been studied in relatively few species of micro-
sporidia. Brooks (1988) reviewed the effects of differ-
ent abiotic factors on the environmental persistence
of microsporidia infecting insects. According to this
review, spore survival could vary from hours to
years, depending on the species and the combination
of abiotic factors to which the spores were subjected.
Relying solely on propagules to persist in a fluc-
tuating environment, however, may prove risky.
Environmental persistence of spores is likely to
trade-off with spore production and/or within-host
growth (Bonhoeffer, Lenski & Ebert, 1996), and thus
reduce parasite competitiveness (Frank, 1996).
During periods of high host density more competi-
tive parasites with less resistant spores could be
favoured, as they will produce more spores.
Similarly, co-infections would lead to within-host
* Corresponding author. Present address : Institut fu
¨
r
Zoologie und Limnologie, Abteilung Ultrastruk-
turforschung und Evolutionsbiologie, Universita
¨
t
Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria.
E-mail: dita.vizoso@uibk.ac.at
501
Parasitology (2005), 130, 501–509. f 2005 Cambridge University Press
doi:10.1017/S0031182004006699 Printed in the United Kingdom
competition, where parasites with low environmental
persistence but high within-host reproduction
could be favoured. Moreover, environmental per-
sistence is limited, with a gradual decay of infectivity
with time. During extended periods of low host
density the parasite might thus be lost from the
system.
The evolution of vertical transmission has been
suggested as another adaptation to reduce the risks
of parasite extinction due to low host densities
(Lucarotti & Andreadis, 1995 ; van Baalen, 2000 ;
Agnew et al. 2002). Vertical transmission (from
parent to offspring) may help a parasite to persist
in a host population even if host density is very low.
Exclusive vertical tr ansmission, however, also bears
risks. If host density increases, the harmful effect of
parasites may lower the competitiveness of infected
hosts relative to that of uninfected hosts, reduce
parasite prevalence and eventually lead to parasite
extinction. For that reason, it has been postulated
that parasites that are transmitted exclusively verti-
cally will either evolve towards mutualism or disap-
pear from the host population (Bull, Molineux &
Rice, 1991 ; Frank, 1991, 1996). Further, vertical
transmission is usually uniparental, and the non-
transmitting sex effectively becomes a dead-end for
vertical transmission. In microsporidia, it has been
suggested that vertical transmission evolved several
times (Smith & Dunn, 1991 ; Dunn & Smith, 2001),
being considered an important adaptation to fluc-
tuating environments in this group of parasites
(Lucarotti & Andreadis, 1995). Most vertically
transmitted microsporidia, however, also transmit
horizontally (Becnel & Andreadis, 1999; Dunn &
Smith, 2001). Mixed routes of transmission could
provide a way of overcoming the constraints imposed
by either route alone (Agnew et al. 2002).
The microsporidium Octosporea bayeri Jı
´
rovec
1936 was first described as a parasite of Daphnia
magna in the Lednice Biological Station, Czech
Republic. Jı
´
rovec (Jı
´
rovec, 1936) described O. bayeri
infecting the fat cells of D. magna, which suffered
hypertrophy due to spore proliferation. O. bayeri
was later found in D. magna populations of the
rock-pools of the Tva
¨
rminne Archipelago in
Southern Finland (Green, 1957) and in Hampton
Court, South of London (Green, 1974). The
Tva
¨
rminne D. magna populations are inherently un-
stable due to the frequent drying up of the rock pools,
followed by sudden invasion of brackish or rain water
(Ebert, Hottinger & Pajunen, 2001; H aag et al.
2002). The prevalence of O. bayeri in this meta-
population, however, is high, with 45% of the popu-
lations containing infected hosts, and an average
prevalence close to 50% within those populations
(Ebert et al. 2001). To achieve high prevalences in
such an unstable environment O. bayeri requires
strategies to survive frequent phases of low host
density and local host extinctions. This study in-
vestigates the infection mechanisms of O. bayeri, and
aims at elucid ating its transmission routes in the
context of some of the environmental constraints this
parasite faces in its natural Daphnia metapopulation.
MATERIALS AND METHODS
System
Isolates of Octosporea bayeri were produced by
collecting single-infected female Daphnia magna
from rock-pool populations in the Tva
¨
rminne
Archipelago, Southern Finland, and allowing them
to reproduce clonally to produce iso-female host
lines. The infection can be maintained in the lab-
oratory in D. magna populations obtained from such
iso-female host lines. Spores can be collected from
dying or dead individuals to infect new host clones
through horizontal transmission. The O. bayeri
isolates were cultured in their original host clones,
or in novel host clones. All cultures were kept in
100–400 ml of artificial medium (Klu
¨
ttgen et al.
1994; modified after Ebert, Zschokke-Rohringer &
Carius (1998)) at 20 xC in a dark : light cycle of
8 : 16 h, and fed regularly with the green algae
Scenedesmus sp. All experiments were done under
these conditions, unless stated otherwise. Table 1
summarizes the experiments performed and their
main results.
Table 1. Summary of experiments and results
Number Experiment Main result
1 Spore-dose Horizontal transmission depends on the density of
propagules present in the medium.
2 Timing of horizontal transmission
and the potential influence of
parasite isolate and host sex
Horizontal transmission only occurs after host death.
Neither host sex nor parasite isolate influenced infectivity.
3 Spores in aqueous solution Spores are viable for at least 24 days outside the host,
in aqueous solution.
4 Desiccation of spores Spores remain viable after at least 5 weeks of desiccation.
5 Transmission to parthenogenetic
offspring
Transmission likely occurs in the ovary.
6 Transmission to sexual offspring Resting, sexual, eggs are infected vertically.
D. B. Vizoso, S. Lass and D. Ebert 502
Pathology of infection and spore morphology
To observe the tissues affected by O. bayeri, infected
female and male D. magna from stock cultures were
isolated and anaesthetized in a dilution of carbonized
water for up to 2 min. The animals were then placed
in a drop of medium on glass slides and observed
using a phase-contrast microscope at 40 to 100r
magnification. Pictures were taken with a FireWire
Video Camera. To observe the spores, whole D.
magna of various ages were placed on glass slides with
a drop of medium. A cover-slip was pressed upon
the Daphnia with a rotating motion so as to spread
the tissues evenly. These wet-mount preparations
were analysed with phase-contrast microscopy, mag-
nification ranging between 300 and 600r. Pictures
of spores were taken from such wet-mount prep-
arations, with a FireWire Video Camera, and with a
Nikon E990. Spores were measured directly from
digital image s using the public domain software
Object-Image2.10 (http://simon.bio.uva.nl/).
Horizontal transmission
Experiment 1. To determine if the rate of horizontal
infection depends on the concentration of spores in
the medium, a dose experiment was performed.
Uninfected D. magna females of 3 days of age were
singly placed in 20 ml of medium containing differ-
ent amounts of O. baye ri spores (0, 10, 100, 1000,
10 000, and 100 000), 12 individuals per spore-dose.
After 4 days 80 ml of medium were added to each
jar, and the Daphnia dissected for observation at
day 18 post-inf ection. The infective status was de-
termined by the presenc e of spores (at 600r).
Experiment 2. To determine when horizontal
transmission occurred, a set of controlled exposures
of uninfected D. magna to hosts infected with 3 iso-
lates of O. bayeri were performed. Juvenile, infected
Daphnia from mass cultures (15 female and 15 male
donors per isolate) were transferred individually into
100 ml of medium, and served as spore donors.
Uninfected female Daphnia of 3 days of age served as
recipients, and were put in contact with the spore
donors at 4 consecutive times. On the day of transfer,
2 recipients were added to each jar (T1). After 6 days,
the recipients were transferred to fresh medium.
The procedure was repeated with a second batch
of recipients, using the same spore donors (now
6 days older, T2). Uninfected recipients were further
added whenever a spore donor died (T3), and
6 days after the spore donors death (T4). For the last
exposure (T4), the body of the dead spore donors
was homogenized and the spore solution was added
to 100 ml of medium. All recipients were kept
in contact with the spore donor (T1-T3) or in the
medium with spores (T4) for 6 days. All exposed
Daphnia were allowed to reproduce clonally, and the
presence of spores was assessed in both mothers
and offspring about 1 month after exposure. To test
for the effect of parasite isolate, donor sex, and time
of exposure on the frequency of infection, the data
were fitted with a Logistic Regression (Kleinbaum
et al. 1998).
Environmental persistence of spores
Experiment 3. Spores in solution. Medium con-
taining a variable amount of dead Daphnia infected
with 3 isolates of O. bayeri was maintained at 20 xCin
100 ml jars, without adding fresh medium. A total
of 22 jars were used (8, 7, and 7 for each isolate
respectively). After 24 days 5 uninfected 4 to
10-day-old female Daphnia were added to the me-
dium. The females were transferred individually to
fresh medium when they matured, and the presence
of spores was determined in the survivors after they
had produced their second clutch of eggs. The effect
of the parasite isolate on the frequency of infection
was tested with the Likelihood Ratio (Kleinbaum
et al. 1998).
Experiment 4. Desiccation of spores. Spore resist-
ance to desiccation was tested by determining the
infectivity of spores after a period of desiccation.
Populations of D. magna infected with combinations
of O. bayeri isolates were kept in 400 ml jars for 4
months. The sediment accumulated dur ing this
period was spread over fil ter paper and allowed to
dry at 20 xC with shaded artificial light during 5
weeks. The sediment was then exposed to continuous
artificial light at 32 xC during 3 days and resuspended
in 100 ml of medium. Ten uninfected Daphnia (ages
3 to 15 days) were added to each of these suspensions.
The presence of spores in all individuals and their
offspring was determined in wet mounts after 4
weeks. A total of 107 replicates (sediment from
independent populations), infected with combi-
nations of 7 different parasite isolates (2–4 replicates
per isolate combination) were used.
Vertical transmission
Experiment 5. Transmission to parthenogenetic
offspring. Mature female Daphnia infected with 1
isolate of O. bayeri were kept individually in 100 ml
of medium and checked 4 times per day to assess
their reproductive state. Daphnia with full ovaries
but empty brood chambers w ere observed every
10 min until new eggs were released into the brood
chamber. The eggs were then carefully flushed out
from the brood cha mber of the living mother using a
Pasteur pipette, and transferred individually into
2
.
5 ml of fresh medium. After eclosion, the Daphnia
were transferred to 100 ml of medium, fed regularly,
and analysed for the presence of spores when they
were 1 month old.
Transmission of Octosporea bayeri 503
Experiment 6. Transmission to sexual offspring.
Sexual eggs (=resting eggs, ephippia) from 126
laboratory populations of Daphnia infected with
combinations of 7 isolates of O. bayeri were accumu-
lated in the cultures’ sediment during 4 months as
described in Exp. 3. Briefly, the ephippia were dried
along with the sediment from the jars at 20 xC during
5 weeks, then exposed to continuous artificial light at
32 xC during 3 days. The ephippia were carefully
washed under running water for 5 min to remove
sediment and spores that might stick to their surface,
and were then placed per population in 50 ml of
medium at 20 xC until hatching. The hatched
Daphnia of each population were kept in the same jar
until they reproduced, thus producing a new popu-
lation that was allowed to grow up to the second
generation. During the growth of the populations
we allowed horizontal transmission, thus increasing
the possibilities of detecting the infection even if only
1 of the hatched Daphnia had carried the infection
through the ephippial phase. The presence of spores
was determined in 10 randomly chosen adults (both
hatched Daph nia and their offspring) from each of
these new populations. Additionally, groups of 5
ephippia from 4 populations were immersed in
diluted commercial bleach for 1, 2, or 5 min, to en-
sure no viable spores remained on the surface. These
groups of ‘ bleached’ ephippia were then grown into
populations as above.
To further determine whether ephippia acquire
the infection vertically or through spores attached in
the surface, embryos were removed from 20 ephippia
from infected populations, and washed carefully in
medium. The embryos were then analysed with a
PCR probe to detect the presence of microsporidian
SSUrDNA as described by Canning et al. (2002).
Briefly, DNA was extracted with the DNeasy Tissue
Kit (Qiagen) and PCR was carried out using the
microsporidian primers Pmp1 (5k-CA CCAGGTT
GATTCTGCCTGAC-3k) and HG4r (5k-TGGTC
CGTGTTTCAAGACGGG-3k) (Zhu et al . 1993 ;
Gatehouse & Malone, 1998).
RESULTS
Pathology of infection and spore morphology
As described by Jı
´
rovec (1936), D. magna infected
with O. bayeri appear opaque, the inner tissues filled
with a pale mass of spores (Fig. 1). In the Daphnia
studied here, the infection was found not only in the
fat cells (Jı
´
rovec, 1936 ; Green, 1957, 1974), but also
in the ovary. This can be observed in whole animals,
as the mass of spores invades the ovaries, making
them opaque (arrow in Fig. 1A). In advanced stages
of infection, the mass of spores spreads through the
entire body cavity (Fig. 1B).
Spores varied in size, shape, and refringency, and
could be classified into 3 morphological types
(Fig. 2). One kind of spore, is oval in shape and
refringent under the phase-contrast microscope
(Fig. 2A), and between 4
.
9 and 5
.
6 mm long and 2
.
2
and 2
.
3 mm wide (321 spores measured in 34 hosts,
2 to 15 spores/host, 8 parasite isolates). Empty shells
of germinated spores of this type can be found in
wet-mount preparations (arrow in Fig. 2A), allowing
the measurement of the spore wall, which was
0
.
49–0
.
53 mm thick (23 spores measured). The thick
spore wall prevents the visualization of the sporo-
plasm using light microscopy. Germination is en-
hanced in wet-mount preparations when pressure
is increased (Fig. 3A). The second spore type shown
in Fig. 2B varies between 4 and 5
.
2 mm in length
and 1
.
6 and 2
.
1 mm in breadth (286 spores measured
in 36 hosts, 1 to 15 spores/host, 8 parasite isolates).
It has a pear-like shape, and the sporoplasm can be
seen in phase-contrast. Its reduced refringency sug-
gests a thinner spore-wall. Germination occurs fre-
quently in wet-mount prepara tions, suggesting that
this spore type actually corresponds to mature spores
and not to early stages of the spore type described
above (Fig. 3B). A third spore type (Fig. 2C), less
Fig. 1. Morphology of infection with Octosporea bayeri.
(A) Mature infected (left) and uninfected (right) Daphnia
magna females. The spores of O. bayeri accumulate in
the fat cells and in the ovary (arrow). (B) An advanced
infection (left) showing the spread of spores throughout
the body cavity of a female D. magna. Uninfected
female (right) shown for comparison.
D. B. Vizoso, S. Lass and D. Ebert 504
frequently observed than the previous spore types,
is of intermediate refringency (spore wall 0
.
35–
0
.
45 mm, 29 spores measured) and elongated in
shape, straight or slightly curved. This kind of
spore varies from 6
.
8to12mm in length, and between
1
.
6 and 2
.
1 mm in breadth (163 spores measured in
22 hosts, 1 to 12 spores/host, 6 parasite isolates).
Germination of this spore type can also be observed
in wet-mount preparations (Fig. 3C), the polar tube
being up to 80 mm long. Although the different spore
types observed in O. bayeri possibly correspond
to typical microsporidian spore types, ultimate tests
of the role of each spore type would require ultra-
structural studies, type-specific separation and in-
fection, assays of environmental persistence, and
intra-host germination.
Horizontal transmission
Unsurprisingly, horizontal transmission of O. bayeri
depends on the concentration of spores present in
the medium, as shown by the results of Exp. 1.
Figure 4 shows the infectivity (percentage of infected
individuals) for each spore-dose (log
10
of number of
spores in 20 ml). A minimum of 100 spores in 20 ml
was required for any infection to occur, and 10 000
spores were enough to acquire 100 % infection.
The results of Exp. 2 suggest that horizontal
transmission of O. bayeri occurs only after host death.
Uninfected Daphnia exposed to living infected hosts
neither developed spores, nor produced infected
offspring (Fig. 5; Exp. 2). Only recipients exposed to
the spore donors within the 6 days after their death
developed infections. The proportion of infections
increased significantly after the dead hosts were
Fig. 2. Spore types of Octosporea bayeri observed with
phase-contrast microscopy. (A) Refringent spores. The
arrow indicates an empty spore shell, probably after
germination. (B) Non-refringent spores. (C) Spores of
intermediate refringency.
Fig. 3. Germinated Octosporea bayeri spores in wet
mount preparations. (A) Germinated type-A spore.
(B) Germinated type-B spores. (C) Germinated type-C
spores of intermediate refringency. Arrows indicate polar
tubes.
Fig. 4. Percentage of Daphnia magna infected when
exposed to different spore-doses of Octosporea bayeri.
The columns represent the infectivity for each spore-
dose, calculated as the log
10
of the total number of spores
added to 20 ml of medium.
Transmission of Octosporea bayeri 505
crushed (Likelihood Ratio Chi-Square=45
.
3,
P<0
.
0001) with no influence of parasite isolate
(Likelihood Ratio Chi-Square=0
.
15, P=0
.
92) or
spore donor sex (Likelihood Ratio Chi-Square=
1
.
33, P=0
.
25).
Environmental persistence of spores
Spores remained infective when kept in aqueous
solution at 20 xC for 24 days, as shown by the results
of Exp. 3. Overall, 36
.
4% of jars containing medium
with spores produced infections. In those jars, all
surviving Daphnia were infected. The parasite isolate
did not have a significant effect on infectivity
(Likelihood Ratio Chi-Square=0
.
32, P=0
.
85).
Further, uninfec ted female Daphnia that were ex-
posed to spores desiccated for 5 weeks and resus-
pended in medium acquired the infection in 54% of
the cases (107 replicates across all parasite isolates,
Exp. 4).
Vertical transmission
In Exp. 5, a total of 79 parthenogenetic eggs removed
from the brood chamber of 27 infected Daphnia
eclosed and survived until spore analysis. All
individuals contained spores, suggesting that vertical
transmission to parthenogenetic offspring is near to
100% efficient, and that the infection occurs before
or during egg release into the brood chamber of the
mother.
Ninety-eight of the 126 replicates (78 % hatching
success) of Exp. 6 produced a new population of
Daphnia. Among those, only 84 % of populations
resulted in infection with O. bayeri. The ephippia
that were immersed in bleach had 67 % hatching
success, and all develop ed into infected females. The
presence of the parasite in the embryos was further
confirmed in 18 of the 20 ephippia analysed with
PCR. Therefore infection to ephippia seems to occur
vertically and not through spores in the sediment
but, in contrast to the vertical transmission to par-
thenogenetic offspring, vertical transmission to
sexual eggs is less than 100% efficient.
Parasite life-cycle
Given the observations on transmission of O. bayeri,
a life-cycle of the parasite in its host can be proposed
(Fig. 6). Horizontal transmission (lower part of
Fig. 6) occurs when infected female or male hosts
die and spores are released to the environment.
Environmental spores survive outside the host for
at least short periods of time in different conditions,
increasing the chances of infecting new hosts even
after brief periods of local extinction of the host. An
infected female can also transmit the parasite to its
parthenogenetic offspring through direct vertical
transmission. This transmission route is maintained
during the parthen ogenetic cycle of D. magna (upper
part of Fig. 6). Vertical transmission also occurs in
the sexual cycle, likely through the mother. Finally,
ephippia may serve as a vehicle for parasite dormancy
and dispersal, with a new cycle of vertical and/or
horizontal transmission starting after hatching.
DISCUSSION
Transmission and persistence
Horizontal transmission of O. bayeri occurred
only after host death. This is characteristic of
microsporidia infecting non-epithelial tissues, where
the release of spores requires some degree of tissue
destruction before the spores can be released (Becnel
& Andreadis, 1999). By the time of death, most in-
fected hosts are filled with spores, as the parasite
grows rapidly inside the host, as long as the host is
still alive (Vizoso & Ebert, 2004). As suggested by the
increase of infectivity when hosts were crushed, the
rupture of spore-containing tissues in D. magna does
Fig. 5. Percentage of infections with three isolates of Octosporea bayeri obtained when exposing young uninfected
Daphnia magna to spore donors at 4 different times (T) of their lives (see text for details). T1 and T2 are separated by 6
days, and the spore donors were alive. At T3 the spore donors had just died, and at T4 the host was crushed after been
dead for at least 6 days. A, B and C represent the 3 parasite isolates, and different greys represent the sex of the spore
donor (dark for female and light for male).
D. B. Vizoso, S. Lass and D. Ebert 506
not occur immediately after host death. In natur e,
this process may be enhanced by bacterial and
detritivore activity. Moreover, D. magna often
browses over the sediment, stirring up particles that
are then ingested by filter feeding (Ebert, 1995), and
may therefore ingest spores present in decaying
infected D. magna.
The survival of spores outside the host, both dry
and in wet conditions, suggests that spores may be
able to persist in the field in the absence of suscep-
tible hosts. This may in turn enhance the perma-
nence and prevalence of O. bayeri in the D . magna
metapopulations. First, spores remained viable in
aqueous solution for at least 1 host generation. The
chances of new infections may thus increase as sus-
ceptible hosts might encounter viable spores even 24
days after the spore donor died. Even if an infected
population would become extinct, susceptible D.
magna that re-colonize the pond may thus acquire the
infection, maintaining the parasite in the pond.
Second, spores accumulated in the sediment re-
mained viable after 5 weeks of desiccation. In the
natural habitat, ponds dry frequently during mid-
summer. In this situation, producing spores resistant
to desiccation may allow O. bayeri to infect hosts that
either hatch from ephippia or invade the rock pool
once rainwater has refilled it. Resistance of spores to
desiccation and light was thought to be absent in
aquatic microsporidia (Becnel & Andreadis, 1999),
and may be a particular adaptation of O. bayeri to the
highly unstable environmental conditions found in
the rock pools of the Finnish metapopulation. It is
also possible that the pre sence of sediment (in our
experiment as well as often in the field) enhances
spore survival, by offering some protection from
light and a source of moisture.
O. bayeri showed a highly efficient vertical trans-
mission to parthenogenetic offspring. In field situ-
ations of low host density and little or no competition
with uninfected hosts, vertical transmission to par-
thenogenetic offspring may allow O. bayeri to remain
in the host population. D. magna hatching from
ephippia were also infected. In the field, this route of
infection may bring two advantages to the parasite.
First, it could provide a way of surviving the extreme
conditions usually present during host diapause.
Infecting ephippia would be a safe way of ensuring
that (at least some) infected hosts will continue
Fig. 6. Suggested life-cycle of Octosporea bayeri infecting Daphnia magna. Grey stippled lines indicate within-host
parasite proliferation. See text for details.
Transmission of Octosporea bayeri 507
the cycle after diapause is over. Relying solely on
the persistence of spores may result in local parasite
extinction, as spores might die or be removed by
wind or water. The thick, melanized coat of the
ephippia may also protect the parasite from the en-
vironmental conditions. The second advantage of
parasitizing ephippia is dispersal. In systems of rock
pools, ephippia can be passively dispersed by water,
birds or wind (Ranta, 1979). Parasitized ephippial
eggs may thus serve as vehicles for parasite dispersal,
potentially spreading the infection throughout the
rock pool system. As the D. magna metapopulation
in which O. baye ri is found is highly dynamic with
20% extinctions per pool per year (Pajunen, 1986)
effective di spersal is essential for persistence in the
host population.
Although our experiments suggest that the per-
sistence of O. bayeri might be high, Ebert et al.
(2001) showed that pools recolonized by Daphnia
are usually free of parasites. O ther ecological factors
that may be limiting parasite persistence in this
system are over-wintering (e.g. resistance to low
temperatures), immigration of resistant clones, and
removal of spores and ephippia by water or wind.
Mechanisms of vertical transmission
The results of Exp. 3 (transmission to partheno-
genetic offspring) suggest that vertical transmission
occurs prior to egg release into the brood chamber.
Although the infection could still occur in the brood
chamber during the first 10 min, the high trans-
mission efficiency and the absence of any horizontal
transmission while the hosts are alive make this
possibility unlikely. The mass of spores observed in
the ovaries further suggests that transmission may
be transovarial. However, eggs could also be infected
in the passage from the ovary to the brood chamber
(transovum infection, Canning, 1982). Conclusive
evidence should include ultrastructural analyses of
infected D. magna ovaries.
The results of Exp. 4 suggest that sexual eggs are
also infected vertically. Infection via spores attached
to the ephippia is unlikely. First, embryos extracted
from the ephippia contai ned copies of the parasite
DNA. Second, the percentage of infections in
ephippia exposed to the sedim ent was considerably
lower than that of the ephippia coming from infected
populations (Exp. 4). Vertical transmission to sexual
offspring through males also seems unlikely, as no
male-to-female infection was observed in Exp. 1, and
no cases of transmission via sperm are known (Becnel
& Andreadis, 1999). The exposed females, however,
might have been too young for mating to occur.
Therefore, experiments that explicitly assess the
possibility of paternal transmission are required.
We cannot compare the efficiencies of the par-
thenogenetic and the sexual transmission with our
experimental set up. However, it seems safe to
conclude that whereas parthenogenetic transmission
in these isolates rarely fails (which has been repeat-
edly corroborated during the keeping of the
cultures), the transmission to sexual offspring is not
as efficient, and offers a way of purging the parasite
from an infected Daphnia clone. Production of
uninfected sexual offspring by infected Daphnia may
be due to differences in the process of parasite
transmission to the egg or embryo, a hypothesis that
requires thorough examination (e.g. through ultra-
structural analyses). It is also possible that the em-
bryo is able to survive the extreme conditions (high
temperatures, drought) whereas the parasite is not.
Another possibility, more exciting but difficult to
test, is that se xual offspring may have acquired
resistance to the maternal parasite strain through
recombination. In both cases, if transmission to
sexual offspring is not perfect, sexual reproduction
could allow infected populations to reduce parasite
prevalence.
In conclusion, our results suggest that both verti-
cal and horizontal transmission are required for the
persistence of O. bayeri under the harsh conditions
the host metapopulation is exposed to, and that
the advantages of each route may compensate the
disadvantages of the other. Vertical transmission
is probably crucial during periods of low host density
and may play an important role in parasite dispersal.
On the other hand, horizontal transmission, through
the release of large quantities of spores that may
boost the parasite’s prevalence in each pond, is
necessary to counteract the negative effects of
between-host competition that would arise due to
vertical transmission.
Some of the pictures were taken in the lab of Nico Michiels
with the help of Lukas Scha
¨
rer. Christoph Haag and
Ju
¨
rgen Hottinger helped with the collection of some of the
samples. Some of the parasite isolates were maintained in
the laboratory by J. Hottinger, Patrick Mucklow, and Marc
Zbinden. J. Hottinger and Lusia Sygnarski provided in-
valuable laboratory assistance. Marc Capaul performed the
dose-experiment. D. B. V. and S. L. further profited from
discussions with D. Refardt and M. Zbinden. DBV re-
ceived financial support from CONICIT (Venezuela) and
the Roche Research Foundation (Switzerland, grant 2002-
111). S. L. acknowledges funding by the German Research
Foundation (DFG, grant LA 1400/1-1). The laboratory
material was financed by the Swiss National Foundation.
REFERENCES
AGNEW, P., BECNEL, J. J., EBERT, D. & MICHALAKIS, Y. (2002).
Symbiosis of microsporidia and insects. In Insect
Symbiosis (ed. Bourtzis, K.), pp. 145–163. CRC Press
LLC, Florida.
ANDERSON, R. M. & MAY, R. M. (1978). Regulation and
stability of host-parasite population interactions.1.
Regulatory processes. Journal of Animal Ecology 47,
219–247.
ANDERSON, R. M. & MAY, R. M. (1981). The population
dynamics of micro-parasites and their invertebrate
D. B. Vizoso, S. Lass and D. Ebert 508
hosts. Philosophical Transactions of the Royal Society
of London, Series B 291, 451–524.
ANDERSON, R. M. & MAY, R. M. (1986). The invasion,
persistence and spread of infectious-diseases within
animal and plant-communities. Philosophical
Transactions of the Royal Society of London,
Series B 314, 533–570.
BECNEL, J. J. & ANDREADIS, T. G. (1999). Microsporidia in
insects. In The Microsporidia and Microsporidiosis
(ed. Wittner, M. & Weiss, L. M.), pp. 447–501.
American Society for Microbiology, Washington, D.C.
BIGLIARDI, E. & SACCHI, L. (2001). Cell biology and
invasion of the Microsporidia. Microbes and Infection 3,
373–379.
BONHOEFFER, S., LENSKI, R. E. & EBERT, D. (1996). The curse
of the pharaoh: The evolution of virulence in pathogens
with long living propagules. Proceedings of the Royal
Society of London, Series B 263, 715–721.
BROOKS, W. M. (1988). Entomogenous protozoa. In CRC
Handbook of Natural Pesticides. Microbial Insecticides,
Part A : Entomogenous Protozoa and Fungi (ed. Ignoffo,
C. M.), pp. 1–150. CRC Press, Boca Raton, Florida.
BULL, J. J., MOLINEUX, I. J. & RICE, W. R. (1991). Selection of
benevolence in a host-parasite system. Evolution 45,
875–882.
CANNING, E. (1982). An evaluation of protozoal
characteristics in relation to biological control of pests.
Parasitology 84, 119–149.
CANNING, E. U., REFARDT, D., VOSSBRINCK, C. R., OKAMURA,
B. & CURRY, A. (2002). New diplokaryotic microsporidia
(Phylum Microsporidia) from freshwater bryozoans
(Bryozoa, Phylactolaemata). European Journal of
Protistology 38, 247–265.
DUNN, A. M. & SMITH, J. E. (2001). Microsporidian life cycles
and diversity : the relationship between virulence and
transmission. Microbes and Infection 3, 381–388.
EBERT, D. (1995). The ecological interactions between a
microsporidian parasite and its host Daphnia magna.
Journal of Animal Ecology 64, 361–369.
EBERT, D., HOTTINGER, J. W. & PAJUNEN, V. I. (2001).
Temporal and spatial dynamics of parasite richness
in a Daphnia metapopulation. Ecology 82, 3417–3434.
EBERT, D., ZSCHOKKE-ROHRINGER, C. D. & CARIUS, H. J.
(1998). Within- and between-population variation for
resistance of Daphnia magna to the bacterial
endoparasite Pasteuria ramosa. Proceedings of the Royal
Society of London, Series B 265, 2127–2134.
FRANK, S. A. (1991). Ecological and genetic models of
host-pathogen coevolution. Heredity 67, 73–83.
FRANK, S. A. (1996). Models of parasite virulence.
Quarterly Review of Biology 71, 37–78.
GATEHOUSE, H. S . & MALONE, L. A. (1998). The ribosomal
RNA gene region of Nosema apis (Microspora) : DNA
sequence for small and large subunit rRNA genes and
evidence of a large tandem repeat unit size. Journal of
Invertebrate Pathology 71, 97–105.
GREEN, J. (1957). Parasites and epibionts of Cladocera in
rock pools of Tva
¨
rminne archipelago. Archivum
Societatis Zoologicae Botanicae Fennicae ‘‘Vanamo ’’
12, 5–12.
GREEN, J. (1974). Parasites and epibionts of Cladocera.
Transactions of the Zoological Society of London 32,
417–515.
HAAG, C. R., HOTTINGER, J. W., RIEK, M. & EBERT, D. (2002).
Strong inbreeding depression in a Daphnia
metapopulation. Evolution 56, 518–526.
JI
´
ROVEC, O. (1936). U
¨
ber einige in Daphnia magna
parasitierende Mikrosporidien. Zoologischer Anzeiger
116, 136–142.
KLEINBAUM, D. G., KUPPER, L. L., MULLER, K. E. & NIZAM, A.
(1998). Applied regression Analysis and Other
Multivariate Methods. Duxbury Press,
Pacific Grove, CA.
KLU¨TTGEN, B., DU¨ LMER, U., ENGELS, M. & RATTE, H . T.
(1994). ADaM, an artificial freshwater for the culture of
zooplankton. Water Research 28, 743–746.
LUCAROTTI, C. J. & ANDREADIS, T. G. (1995). Reproductive
strategies and adaptations for survival among obligatory
microsporidian and fungal parasites of mosquitoes : a
comparative analysis of Amblyospora and Coelomyces.
Journal of the American Mosquito Control Association 11,
111–121.
MADDOX, J. V. (2002). Environmental persistence of
Microsporidia. In Factors Affecting the Survival of
Entomopathogens (ed. Baur, M. E. & Fuxa, J. R.).
Southern Cooperative Series Bulletin 400. Available at :
http://www.agctr.lsu.edu/s265/default.htm
ONSTAD, D. W., MADDOX, J. V., COX, D. J. & KORNKVEN,
E. A. (1990). Spatial and temporal dynamics of
animals and the host-density threshold in
epizootiology. Journal of Invertebrate Pathology
55, 76–84.
PAJUNEN, V. I. (1986). Distributional dynamics of Daphnia
species in a rock-pool environment. Annales Zoologici
Fennici 23, 131–140.
RANTA, E. (1979). Niche of Daphnia species in rock pools.
Archiv fu
¨
r Hydrobiologie 87, 205–223.
REGOES, R. R., HOTTINGER, J. W., SYGNARSKI, L. & EBERT, D.
(2003). The infection rate of Daphnia magna by Pasteuria
ramosa conforms with the mass-action principle.
Epidemiology and Infection 131, 957–966.
SAUVAGE, F., LANGLAIS, M., YOCCOZ, N. G. & PONTIER, D.
(2003). Modelling hantavirus in fluctuating populations
of bank voles : the role of indirect transmission on virus
persistence. Journal of Animal Ecology 72, 1–13.
SMITH, J. E. & DUNN, A. M. (1991). Transovarial
transmission. Parasitology Today 7, 146–148.
VAN BAALEN, M. (2000). Parent-to-offspring infection and
the struggle for transmission. In Evolutionary Biology of
Host–Parasite Relationships: Theory Meets Reality (ed.
Poulin, R., Moran, S. & Skorping, A.), pp. 97–116.
Elsevier, Amsterdam.
VIZOSO, D. B. & EBERT, D. (2004). Within-host dynamics
of a microsporidium with horizontal and vertical
transmission: Octosporea bayeri in Daphnia magna.
Parasitology 128, 31–38.
ZHU, X., WITTNER, M., TANOWITZ, H. B., CALI, A. & WEISS,
L. M. (1993). Nucleotide sequence of the small ribosomal
RNA of Encephalitozoon cuniculi. Nucleic Acids Research
21, 1315.
Transmission of Octosporea bayeri 509
