MALARIA-INDUCED APOPTOSIS IN MOSQUITO OVARIES : A MECHANISM TO CONTROL VECTOR EGG PRODUCTION
1
Centre for Applied Entomology and Parasitology, Keele University,
Staffordshire ST5 5BG, UK
2
School of Biological Sciences, University of Manchester, 2.205 Stopford
Building, Oxford Road, Manchester M13 9PT, UK
3
School of Life Sciences, Keele University, Staffordshire ST5 5BG,
UK
*
Author for correspondence (e-mail:
h.hurd{at}keele.ac.uk
)
Accepted May 29, 2001
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Summary |
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Key words: mosquito, Anopheles stephensi, oogenesis, follicle resorption, apoptosis, caspase, Plasmodium yoelii nigeriensis, malaria, follicular epithelium, nurse cell
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Introduction |
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Malaria is regarded as the most serious of the parasitic diseases, causing
300-500 million clinical cases and 1.5-2.7 million deaths annually
(www.who.org). It is caused by protozoans of the genus Plasmodium and
is transmitted by anopheline mosquitoes during blood feeding. Within 24h of
ingestion, Plasmodium spp. gametes are fertilised in the blood bolus
within the mosquito midgut, and motile ookinetes, developed from zygotes,
begin to traverse the midgut wall. They transform into oocysts below the basal
lamina of the midgut epithelium (Beier,
1998). These oocysts produce
sporozoites that invade the salivary gland, ready to infect another vertebrate
host during the next blood meal. These developmental stages of the rodent
malaria Plasmodium yoelii nigeriensis cause a significant reduction
in the number of eggs produced by Anopheles stephensi and
Anopheles gambiae following successive blood meals (Hogg and Hurd,
1995a
; Hogg and Hurd,
1995b
; Ahmed et al.,
1999
). Thus, the impact of
infection upon mosquito reproductive fitness is cumulative.
Mosquito meroistic ovaries contain a series of approximately 50 ovarioles
that radiate from a calyx. Each ovariole consists of a germarium and two
follicles or egg chambers. The latter are composed of one oocyte and seven
nurse cells and are enclosed in a follicular epithelium (Sokolova,
1994). Egg production is
cyclical and synchronous. A blood meal initiates the maturation of the
proximate follicles, resulting in oviposition of an egg batch at the end of a
gonotrophic cycle. During this process, the follicular epithelial cells become
patent and separate to allow passage of yolk protein precursors, including
vitellogenin, into the oolemma, where it is internalised by receptor-mediated
endocytosis and deposited in a crystalline form, vitellin (Snigirevskaya et
al., 1997
; Raikhel and
Snigirevskaya, 1998
). In
Anopheles stephensi, yolk protein accumulates in the terminal
follicles from trace levels at 4h post-bloodmeal to a maximum in 2 days (Hogg
et al., 1997
). Nurse cells
synthesise RNA and other macromolecules that are transported to the oocyte
cytoplasm via intercellular canals. By 48-50h, oocyte maturation is
completed; epithelial cells have secreted a chorion (egg shell) and the nurse
cells degenerate. Events surrounding the degeneration of the nurse cells and
follicular epithelium are not well-documented.
When a female mosquito feeds on host blood containing malaria gametocytes,
the gonotrophic cycle begins normally. However, soon after vitellin begins to
accumulate in the terminal follicles, a significant proportion of them undergo
resorption (Carwardine and Hurd,
1997). This process reduces the
size of the final egg batch produced and is the major contributory factor to
Plasmodium-induced fecundity reduction. To understand the
parasite-induced signals that initiate and control fecundity reduction, we are
studying the process of follicle resorption in infected mosquitoes.
The signalling mechanisms and cellular processes involved in
uninfected-insect follicle resorption are poorly understood. Nevertheless, we
thought it likely that malfunctioning cells of the follicular epithelium might
be involved in initiating the process because they play a key role in the
passage of vitellogenin into the oocyte in mosquitoes. We have designed
experiments to investigate this hypothesis. Alternatively, follicular
epithelial cells could play an active part in resorption by enzyme degradation
and phagocytosis of accumulating yolk, as seen in locust resorbing follicles
(Lüsis,
1963). Here, we report that
malaria infection induces apoptosis in cells of the follicular epithelium and
that this programmed cell death results in follicle resorption.
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Materials and methods |
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Male CD mice were infected with Plasmodium yoelii nigeriensis
strain N67 following one serial passage of blood from mice infected from
cryopreserved stocks (Hogg and Hurd,
1995b). Parasitaemia and
gametocytaemia were monitored in Giemsa-stained, thin blood films and
unstained, fresh, thick blood films, respectively, at 24-30h
post-infection.
Nulliparous female mosquitoes (6-8 days post-emergence) were allocated
randomly to two groups and starved for 12-18 h before feeding on anaesthetized
non-infected mice or infected mice (gametocytaemic and exflagellating) 24-30h
post-infection (Jahan and Hurd,
1998). The packed cell volumes
of control and infected mice (litter siblings) were measured immediately prior
to blood-feeding the mosquitoes. The control mouse was selected to have a
packed cell volume within 2% of that of the infected mouse to ensure that
bloodmeal haemoglobin content was not reduced in infected mosquitoes (Taylor
and Hurd, 2001
). For each
study, a group of engorged females that had fed on the infected mouse was
separated and retained to check for prevalence and intensity of infection by
monitoring oocysts developing on the midgut at day 7 post-bloodmeal.
Prevalence of infection always exceeded 90%, and oocyst burden was always over
50 per mosquito (data not shown). The wing length of each mosquito used in the
study was measured as an assessment of mosquito size (Jahan and Hurd,
1997
). No significant
differences between control and infected groups in individual experiments were
found (data not presented).
Follicle resorption and apoptosis in whole ovaries
Fully engorged female mosquitoes were selected at random and assigned to
groups for examination at 16, 18, 20, 22, 24 and 36h post-bloodmeal. All
groups were maintained in the insectary conditions described above and chilled
for 2-3 min at -20°C prior to dissection. Ovaries were dissected in
Aedes physiological saline (APS) (150 mmoll-1 NaCl, 4
mmoll-1 KCl, 1 mmoll-1 CaCl2, 0.1
mmoll-1 NaHCO3, 0.6 mmoll-1 MgCl2
buffered with 25 mmoll-1 Hepes) and dipped in Neutral Red [Gurr,
London; 0.5% (w:v) solution in citrate-phosphate buffer: 0.1 moll-1
citric acid/0.1 moll-1 sodium citrate] at pH 6 for 1 min to assist
the visualization of follicles (Clements and Boocock,
1984). Neutral Red
concentrates in lysosomes and yields a deep red colour at a slightly acid pH
(Wilson, 1990
). Resorbing
follicles exhibit an increased uptake of Neutral Red (Bell and Bohm,
1975
; Clements and Boocock,
1984
). Ovaries were further
dissected into individual ovarioles and examined under a dissecting microscope
at x100 (Carwardine and Hurd,
1997
). The number of resorbing
follicles was recorded. Acridine Orange (50 µmoll-1 in APS) was
then added to the ovaries, and they were immediately examined using
fluorescence filters. The number of follicles containing epithelial cells with
condensed nuclei was counted. Acridine Orange is one of several nucleic acid
stains routinely used to reveal the condensed chromatin characteristic of
apoptotic cells (e.g. McGahon et al.,
1995
; Longthorne and Williams,
1997
).
Detection of DNA fragmentation in ovarian tissue
Apoptotic nuclei were further identified in paraffin-wax-embedded sections
of ovaries by in situ terminal-deoxynucleotidyl-transferase-mediated
dUTP-biotin nick end labelling (TUNEL) using the Oncogene Research Products
TdT-FragEL DNA fragmentation detection kit. The procedure was carried out
according to the manufacturer's instructions, and sections were counterstained
with Methyl Green to aid the morphological evaluation and characterization of
normal and apoptotic cells. Mosquitoes were infected and treated as outlined
above. At each time point, ovaries were dissected in APS then immediately
dehydrated in a graded ethanol series prior to embedding in wax. The objective
here was to confirm that cells of the follicular epithelium were undergoing
apoptosis and to check whether nurse cells, which were not visible in
whole-mounts, were also apoptotic. The TUNEL technique identifies the presence
of DNA fragmentation, an early stage in the process of programmed cell death.
In total, 50 sections were observed, and attention was focused on ovaries from
infected mosquitoes during the early stages of oogenesis, although some
sections from uninfected mosquitoes were examined. The sampling technique thus
precluded statistical comparisons between time points or between infected and
uninfected females.
Ultrastructural examination of ovaries from infected mosquitoes
Ovaries from uninfected and infected females (treated as above) were
dissected in APS and fixed in 3% (v:v) glutaraldehyde in 0.1 moll-1
sodium phosphate buffer, pH 7.4, and post-fixed in 1% (w:v) osmium tetroxide
in the same buffer for 1h before dehydration in a graded ethanol series and
embedding in Spurr's resin. Ultrathin sections were stained with uranyl
acetate and lead citrate and observed with a Joel JEM 100CX11 transmission
electron microscope. Grids from each time point were examined at a series of
magnifications from x1900 to x7200.
Inhibition of apoptosis
To establish conclusively that malaria-induced apoptosis caused follicle
resorption, we attempted to inhibit the activation of apoptosis, using a
caspase inhibitor [z-VAD.fmk, benzyloxycarbonyl-Val-Ala-Asp (Ome)
fluoromethylketone; Garcia-Calvo et al.,
1998; Rodriguez et al.,
1996
; Jacobson et al.,
1996
]. Immediately after blood
feeding on a gametocytaemic mouse, fully engorged females were injected with
0.25 µl of z-VAD.fmk [0.5 mmoll-1 in a solution of dimethyl
sulphoxide (DMSO) 1:100 (v:v) in APS], with DMSO [1:100 (v:v) APS] or with APS
alone. Solutions were injected into the thorax just below the base of a wing
using a finely drawn out, calibrated glass microcapillary tube. A further
control group of uninjected, blood-fed mosquitoes was maintained under
identical conditions. All mosquitoes emerged on the same day and were fed on
the same infected mouse.
Mosquitoes were returned to the insectary and given access to sugar solution (as above) for 24h. At this time, a comparison of the number of resorbing follicles in ovaries from mosquitoes subjected to each treatment was made.
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Results |
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Acridine Orange staining of the same ovaries demonstrated that a similar proportion of follicles contained cells that were undergoing apoptosis to those that were resorbing (see Fig. 3A). A two-way analysis of variance (ANOVA), carried out on arcsine-transformed data, showed an overall increase in the number of resorbing follicles in infected versus uninfected mosquitoes (F1,108=222.25; P<0.001) and an overall increase in apoptosis in follicles from infected compared with control mosquitoes (F1,108=199.10; P<0.001).
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A Tukey's unplanned comparison between pairs of means demonstrated that a significant difference occurred between resorption and apoptosis only in infected females at 36h postfeeding (P<0.05). The reduction in the occurrence of apoptosis at 36h (Fig. 1) was to be expected if apoptosis, which is a short-lived event, occurred prior to follicle resorption. That there is a relationship between resorption and apoptosis is shown by a three-way ANOVA on percentage resorption using a general linear model with percentage apoptosis as covariate, which indicates a significant relationship between the two (F1,96=32.50; P<0.001) irrespective of whether the mosquitoes are infected or uninfected, except at 36h. However, as stated above, by this time most of the apoptosis has already occurred (Fig. 2).
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Apoptosis occurred in the cells of the follicular epithelium
Patterns of nuclei stained with Acridine Orange suggested that apoptosis
was occurring in follicular epithelial cells
(Fig. 3A). We confirmed this by
examination of ultrathin sections of ovaries from infected females. Condensed
nuclear chromatin, a characteristic of apoptosis, was seen in adjacent cells
of the follicular epithelium of many terminal follicles of ovarioles dissected
at the same time points post-bloodmeal as were used for Acridine Orange
staining. No development of patency had occurred in these apoptotic cells by
22h post-bloodmeal, and normal yolk spheres were not evident in oocytes
surrounded by apoptotic cells (Fig.
3C). It appears that the occurrence of a patch of cells undergoing
apoptosis may prevent vitellogenin gaining access to the adjacent oolemma.
DNA fragmentation was detected very early in the infection
process
The initial stage of apoptosis is characterised by DNA fragmentation, an
early event, that is sometimes detectable before the production of gross
apoptotic morphology. Using TUNEL, we were able to detect the onset of
apoptosis in patches of follicular epithelial cells in ovaries from infected
females 16 and 18 h post-bloodmeal. We also observed that some labelled
follicles contained nurse cells with apoptotic nuclei at these times
(Fig. 3B). Fragmentation of DNA
was not detected in the follicles examined at 22, 24 and 36h post-bloodmeal.
This aspect of the study was designed to confirm that the Acridine Orange
staining we had previously observed demonstrated that apoptosis was taking
place in the follicular epithelium. It also enabled us to examine more tissue
than was feasible using electron microscopy because each section contained
several follicles. No quantitative comparison between uninfected and infected
ovaries was undertaken using TUNEL because of our choice of sampling method,
and we did not observe any staining in the few sections from uninfected
mosquitoes that were examined, probably because of the low frequency of
apoptotic follicles in uninfected mosquitoes. It is possible that DNA
fragmentation is completed by 22h or that, because of the low frequency of
follicles with cells in this early stage, they were not included in our
sample.
Caspase inhibition prevents follicle resorption
Administration of the caspase-inhibitor z-VAD.fmk to infected mosquitoes
significantly reduced the proportion of follicles undergoing resorption at 24h
post-bloodmeal from 16%, in untreated females, to 10%. Ovaries from untreated
females had a similar number of resorbing follicles to those in previous
experiments, and injection of the inhibitor-solvent or saline alone did not
reduce this proportion (Fig.
4).
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Discussion |
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In our studies of malaria-infected mosquitoes, females are mated before
they are infected, and thus an indirect effect via changes in the
transfer of, or response to, sex peptides will not be taking place. Nor do we
think that nutrient stress resulting from infection is the prime cause. Using
our experimental regime, no decrease in haemoglobin intake occurs in
mosquitoes feeding on infectious mice (Jahan et al.,
1999; Taylor and Hurd,
2001
), and the protein content
of the blood meal is digested normally (Jahan et al.,
1999
). In addition, follicle
resorption begins at a time when ookinetes are invading the midgut, and
nutrient competition between parasite and host is probably not occurring so
early in infection. As part of our investigations to determine whether there
is a mechanism controlling oogenesis that is unique to infection, or whether
the malaria parasite reduces egg production via a route associated
with other physiological or environmental triggers, we need to understand the
process of follicle resorption in mosquitoes.
We have now established that the programmed cell death of large patches of follicular epithelial cells is an early event in malaria-induced resorption of developing follicles and that apoptosis precedes resorption. Although we observed DNA fragmentation occurring in some of the nurse cells in resorbing follicles, the relative role of these cells is unclear. Our observation that follicular atresia is also associated with apoptosis in a second malaria-infected mosquito species, Anopheles gambiae (data not shown), suggests that this may be a universal response to malaria infection.
The occurrence of apoptosis in mosquito tissue has only recently been
recorded. It was observed in studies of the midgut cells during metamorphosis
(Nishiura and Smouse, 2000)
and malaria ookinete invasion (Han et al.,
2000
), but no previous report
of a role for programmed cell death in any aspect of mosquito oogenesis has
been made. This is in contrast to D. melanogaster, in which studies
of oogenesis are much more advanced. Apoptosis has been reported to occur in
D. melanogaster nurse cells following cytoplasmic dumping, and the
expression of D. melanogaster inhibitors of apoptosis, DIAP1 and
DIAP2, in nurse cells ceases during stages 7 and 8 (Foley and Cooley,
1998
). Soller et al. (Soller
et al., 1999
) suggested that
regulatory switches for oogenesis reside in the ovary and operate at stage
9-10, which may be an apoptosis-sensitive window. Apoptosis of nurse cell
nuclei occurred early in this process, but no Acridine Orange staining of the
follicle cell nuclei occurred nor did they label with TUNEL (Soller et al.,
1999
). However, clones of
defective follicle cells cause degeneration of the whole follicle (M. Bownes,
personal communication). Thus, different processes may occur in D.
melanogaster nurse and follicular epithelium cells. Furthermore,
apoptosis in D. melanogaster follicle cells is induced by ectopic
expression of reaper and hid transgenes and can be
suppressed by co-expression of the baculovirus p35 protein. In these
experiments, substantial death of follicle cells resulted in germline death
(Cho and Nagoshi, 1999
).
Our observation of malaria-induced resorption of anopheline mosquito
follicles has demonstrated that nuclear disintegration and chromatin
condensation occur in large groups of follicular epithelial cells as well as
in some nurse cells. The process of follicle resorption and the role of
apoptosis in normal oogenesis may, therefore, differ between D.
melanogaster and mosquitoes. In particular, there is no single stage
during mosquito oogenesis at which follicle resorption occurs (Clements and
Boocock, 1984), and follicular
epithelial cells may play a more prominent role than nurse cells.
The caspases (Alnemri et al.,
1996) are the biochemical core
of the apoptosis mechanism. They are site-specific proteases acting in a
cascade to bring about the subcellular changes characteristic of apoptosis
(e.g. DNA fragmentation, chromosomal condensation and stimulation of
phagocytosis; Hengartner,
2000
). At least five caspases
have been identified so far in D. melanogaster. The caspase-encoding
genes dcp-1 and decay are involved in D.
melanogaster nurse cell apoptosis, which occurs during normal oogenesis
(Dorstyn et al., 2000
; McCall
and Steller, 1998
). Our work
demonstrates that caspases are involved in mosquito follicle cell apoptosis
and that inhibition of caspase activity stops resorption. This supports our
premise that programmed cell death of follicular epithelial cells initiates
resorption. However, at this stage, we do not know whether the D.
melanogaster decay-like gene is involved.
Ovaries in Plasmodium-infected mosquitoes are responding to the
presence of resorption-inducing signals that may, or may not, be specific to
this infection. The nature of these signals is, however, currently unknown.
There is evidence from the literature that the hormonal milieu influences
insect follicle maintenance/degeneration involving apoptosis (Capella and
Hartfelder, 1998). The balance
between the insect steroid hormone ecdysone (secreted by mosquito ovaries) and
juvenile hormone may be important, as in D. melanogaster. Soller and
colleagues (Soller et al.,
1999
) proposed a model for
oocyte maturation in D. melanogaster that identifies the balance
between two hormones, 20-hydroxyecdysone and juvenile hormone, as playing a
key role. Sex-peptide, transferred to females during mating, inhibits the
apoptosis-inducing effect of 20-hydroxyecdysone during early vitellogenesis
and prevents resorption at this time. Treatment of D. melanogaster
with 20-hydroxyecdysone resulted in the resorption of some egg chambers. This
particular hormonal balance may not, however, be important in mosquitoes. In
uninfected mosquitoes, ecdysteroid titres peak midway through the gonotrophic
cycle, when juvenile hormone levels are low (for a review, see Clements,
1992
). Thus, high ecdysone/low
juvenile hormone titres in the haemolymph may not initiate
apoptosis/resorption. Instead, changes in local hormone synthesis by
individual follicles may be involved in mosquitoes. At present, we do not know
whether infection alters hormone titres, but there is some evidence that the
endocrine system is a possible signalling mechanism between
Plasmodium and mosquito tissue (Ysikevich and Zvantsov,
1999
), and this may have a
role in regulating programmed cell death.
Throughout the initial and subsequent gonotrophic cycles post-infection,
yolk protein accumulates in the haemolymph of infected mosquitoes as the rate
of uptake by the ovaries declines as a result of follicle resorption (Hogg et
al., 1997; Ahmed et al.,
2001
). In addition, infection
results in a significant reduction in the abundance of vitellogenin mRNA that
is likely to result in a reduction in vitellogenin synthesis (Ahmed et al.,
2001
). In infected females,
resources that under normal circumstances would have been devoted to
reproduction are being diverted away from egg production. This is a common
parasite strategy that may exploit a trade-off between egg production and
longevity by manipulation of host resource management (Koella,
1999
). An understanding of the
underlying physiological mechanisms and the fitness advantages to parasite and
vector will help to clarify the adaptive nature and significance of host
fecundity reduction (Read,
1990
; Hurd and Webb,
1997
; Hurd,
2001
).
We have now shown that apoptosis is an important feature of
malaria/mosquito interactions because it seems to precipitate the process that
results in fecundity reduction. This exploitation of the physiological
mechanisms of programmed cell death appears to result from a signal generated
by the parasite, directly or indirectly, as it invades the midgut and
stimulates an immune response (Richman et al.,
1997; Luckart et al., 1998).
This signal may be long-lasting, thus affecting subsequent cycles of egg
production, or new signals, of the same or different origin, may occur at
later stages of infection. Further investigation of this phenomenon should
produce a greater understanding of the diverse factors controlling malaria
transmission via mosquitoes and shed light on the normal processes
involved in the regulation of mosquito egg production.
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Acknowledgments |
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