1
Gregarine infection accelerates larval development of the cat
flea Ctenocephalides felis (Bouché)
M. E. ALARCÓN 1 , A. JARA-F. 2 , R. C. BRIONES 3 , A. K. DUBEY 4 * and
C. H. SLAMOVITS 5
1
Liceo Alemán del Verbo Divino Lynch 350 Los Ángeles, Bío-Bío, Chile
Doctorado en Sistemática y Biodiversidad, Departamento Zoología, Facultad de Ciencias Naturales y Oceanográficas,
Universidad de Concepción, Concepción, Chile
3
División Manejo Ecosistémico, Programa Conservación de Fauna, Bioforest S.A. Concepción, Chile
4
Forest Entomology Division, Forest Research Institute, Dehradun, India
5
Canadian Institute for Advanced Research and Department of Biochemistry and Molecular Biology, Dalhousie University,
Halifax, Nova Scotia, Canada
2
(Received 22 July 2016; revised 12 October 2016; accepted 21 October 2016)
SUMMARY
A high degree of specialization between host and parasite is a well-known outcome of a long history of coevolution, and it is
strikingly illustrated in a coordination of their life cycles. In some cases, the arms race ensued at the establishment of a
symbiotic relationship results in the adoption of manipulative strategies by the parasite. We have already learned that
Steinina ctenocephali, a gregarine living in the alimentary canal of cat flea, Ctenocephalides felis follows its phenology
and metamorphosis. Despite these findings the outcome of their symbiotic partnership (mutualist, parasitic or commensal)
remains unclear. To address this important question, we measured life history parameters of the flea in the presence of
varying infection intensities of gregarine oocysts in laboratory conditions. We found that neither the emergence nor survival rate of fleas was affected by harbouring the gregarines. More surprisingly, our results show that flea larvae infected
with gregarines developed faster and emerged earlier than the control group. This gregarine therefore joins the selected
group of protists that can modify physiological host traits and provides not only new model taxa to be explored in an evolutionary scenario, but also potential development of control strategies of cat flea.
Key words: Ctenocephalides felis, Steinina ctenocephali, oocyst concentration, survival curve, flea development.
INTRODUCTION
The domestic cat flea, Ctenocephalides felis (Bouché)
is the most abundant ectoparasite of cats and dogs
worldwide. The fleas have shown a propensity to
develop resistance to insecticides (Bossard et al.
2002), thus becoming arduous to manage and
control. In addition to developing new chemical
compounds with anti-ectoparasitic activity, other
potential avenues to explore include the development of biological control using natural parasites of
the flea (Henderson et al. 1995).
The gregarine protist, Steinina ctenocephali (Ross,
1909) (Phylum Apicomplexa) inhabits in the alimentary canal of the cat flea. It colonizes in a newly
hatched larva by entry of sporozoites released from
ingested oocysts along with the flea feces. The S. ctenocephali develop intracellularly during larval and
pupal stages of cat flea, once the cat fleas emerge
(as fully grown adult) and start feeding blood, the
gregarine move out to lumen of flea gut attaching
itself in the intestinal wall, and continues feeding
as a trophozoite (Fig. 1A), thus becoming gamonts,
* Corresponding author: Forest Entomology Division,
Forest Research Institute, New Forest, Dehradun,
Uttarakhand, India. E-mail: [email protected]
which undergo syzygy and secrete a gametocyst
wall. The gregarine then produces infective oocysts
(Fig. 1D), which are released and propagated via
fecal discharge. The fecal matter carrying oocysts
also contains undigested blood meal, which serves
as food to the flea juveniles, whilst ensuring transmission and perpetuation of the gregarine (Alarcón
et al. 2011). A wealth of recent work on S. ctenocephali conducted on cat flea populations from
Taiwan have addressed diverse topics such as
dynamic of infection patterns, life history, morphology, taxonomy and molecular systematics of this
gregarine (Alarcón et al. 2011, 2013). Interestingly,
in such studies S. ctenocephali was the only species
of gregarine found associated with this holometabolous insect, which is consistent with the high specificity and monoxenicity usually found among these
apicomplexans (Rueckert et al. 2010). Despite
passing unnoticed since its description in 1909,
recent advances in microscopy and genetics have
greatly facilitated the rediscovery of S. ctenocephali
and allowed investigations leading to new insights
into this organism and its role in the partnership
with the cat flea. An important outcome of the
recent studies is the realization that flea and gregarine shaped a close relationship over time (Alarcón
Parasitology, Page 1 of 7. © Cambridge University Press 2017
doi:10.1017/S0031182016002122
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M. E. Alarcón and others
2
Fig. 1. Cat flea, Ctenocephalides felis infected with Steinina ctenocephali symbionts. (A) Live female cat flea infected with
S. ctenocephali trophozoites observed under stereo zoom microscope (white arrow heads indicate 4 young trophozoites
attached to the intestine wall). (B) Live positive artificially infected male flea. A newly emerged flea after feeding blood of
cat for 4 days revealed the presence of several freshly/newly transformed sporozoites into early trophozoites (white arrow
heads). (C) Scanning electron micrographs (SEM) of trophozoites of S. ctenocephali, partially embedded in the microvilli
(MV) brush of cat flea intestinal epithelium. (D) (SEM), lemon-shaped oocysts (arrowheads) were released from ruptured
gametocyst (RG). (E) Two lemon-shaped S. ctenocephali oocysts observed from gregarine infected flea feces visualized on a
hematocytometer (Hausser Scientific, Horsham).
et al. 2011). The gregarine may decrease fitness of
cat flea, which may explain the unusually low
prevalence of the gregarine in fleas of urban cat
and dog populations (Alarcón et al. 2013), but it
is unclear how the outcome of this microbe–host
interaction affect each partner. Knowing whether
the interaction is beneficial (i.e. mutualism),
harmful (i.e. parasitic) or without effect on cat flea
(i.e. commensalism) will be critical to determine
the viability of a biological control programme
based on gregarines. In order to investigate this
key question, we analysed the effects of S. ctenocephali infection on the survival and development of
the cat flea by experimentally manipulating the
doses of sporozoites ingested by flea larvae, and
monitored the outcome in terms of survival and
length of developmental stages. Here, we have
shown for the first time that S. ctenocephali rather
than negatively affecting the fitness of its host, S.
ctenocephali does not change the survival rate of
the flea. In addition, we found that S. ctenocephali
is capable of manipulating its host life history by
accelerating larval development.
MATERIALS AND METHODS
Flea collection
Adult infected cat fleas, C. felis were collected from
933 dogs and 197 cats in Taipei Animal Shelter,
Taiwan. Fleas were obtained by combing the fur of
each cat and dog with a fine flea comb for 10 min.
Collected fleas were transferred into plastic tubes,
labelled with collection data and maintained individually from each dog and cat, and then taken to
the laboratory in the Division of Entomology,
National Taiwan University, Taiwan. The whole
fleas were prepared as saline solution-mounted
slides and quickly examined individually by using
a dissecting stereo zoom microscope (Leica Zoom
2000, New York) to confirm S. ctenocephali infection. Finally, gregarine infected fleas were separated
and placed into microcells.
Microcells and oocyst collection
Microcells techniques were those used by Thomas
et al. (1996); Hsu and Wu (2000); Rust et al.
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Gregarines effect on cat flea larvae development
3
Fig. 2. Survival curves for cat fleas, exposed to different oocyst concentration. Dose-dependent effect of oocysts on the
survival rate of cat flea observed for an experimental duration of 25 days. (black – 50), (pink – 25), (green –12·5), (blue –
6·25 oocysts mg−1 and (light blue – control).
(2001), and McCoy et al. (2008). These authors had
used basically bandaging-stockinette to prevent the
chamber from being dislodged from experimental
cats. For this purpose, we modified a commercially
available adjustable cat backpack harness, and
adapted it as a jacket (Suppl. figure 1, suppl. video 1).
Microcells were fitted under the jacket and hold in
place by a rubber bracelet. This design is much more
efficient and easy to operate, and, moreover,
maximum of 12 microcells could be placed simultaneously. Fleas were kept into microcells until gametocysts were dehisced. Under stereomicroscope the
absence of gametocyst(s) in the gut was used as indicator that gametocyst(s) had been ruptured and emptied
(releasing oocysts) along with the flea feces excreted.
Flea feces were removed from the microcells and
stored to 4 °C until experiments.
Oocyst manipulation
The concentration of oocysts in feces was determined
with a hemacytometer (Bright-Line, Hausser Scientific, Horsham, PA) under BH2 phase contrast
microscope (Olympus, Tokyo, Japan) (Fig. 1E).
[0·032 gr of feces had a concentration of 270
oocysts/0·001 g (total infective powder, TIP)]. An
initial stock of feces containing 100 oocysts mg−1
was prepared (stock infective powder, SIP) from
TIP by mixing with porcine blood curd powder
(PBC) as diluting solvent. Noticeably, the gregarine-free PBC is an artificial flea larval diet used successfully in the laboratory for optimum full-flea
development (Hsu, 2000; Hsu et al. 2002). From
SIP and following the procedure described
above, we prepared oocyst doses of 50, 25, 12·5,
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M. E. Alarcón and others
4
Table 1. Effects of Steinina ctenocephali on survival and emergence rates of Ctenocephalides felis (±S.E. after
three replications).
Survival (%)
Oocysts dose
(oocysts mg−1)
L1
L2
L3
P
Emergence rate (%)
50
25
12·5
6·25
0·00
89·58 ± 2·08
93·75 ± 0·00
100·00 ± 0·00
95·83 ± 2·08
95·83 ± 4·17
89·58 ± 2·08
93·75 ± 0·00
100·00 ± 0·00
95·83 ± 2·08
95·83 ± 4·17
89·58 ± 2·08
91·67 ± 2·08
100·00 ± 0·00
91·67 ± 4·17
95·83 ± 4·17
77·08 ± 4·17
83·33 ± 2·08
91·67 ± 8·33
77·08 ± 5·51
85·42 ± 11·60
77·08 ± 4·17
83·33 ± 2·08
91·67 ± 8·33
77·08 ± 5·51
87·50 ± 9·55
L1, first instar larvae; L2, second instar larvae; L3, third instar larvae; P, pupa.
6·25 oocysts mg−1 and a control only containing
PBC free of oocysts.
Flea infection and data recording
The uninfected larvae were obtained from a colony
of fleas, which is maintained on cats as hosts, as
described by Hsu and Wu (2000). To infect flea
larvae artificially with S. ctenocephali, we used only
80 wells from a 96-well round-bottom polypropylene tissue culture plates (Costar 3599, Corning Inc.,
Corning, NY). A total of 16 wells per treatment were
loaded individually with 1 mg of infective powder
containing 50, 25, 12·5 and 6·25 oocysts mg−1, with
a control group included. A single 1-day-old flea
egg was placed in each well. The hatching larvae
fed on the powder until emergence as adults. A
daily record of life history parameters (mortality,
instars duration, pupation time, etc.) in each microwell was recorded. To verify gregarine infection,
newly emerged fleas (Fig. 1B) were blood fed
on cat by 3–5 days using microcells. The experiment
was replicated three times, and observations
were documented for each replication separately.
Although, pseudoreplication is a limitation in an
experimental design we used a pseudoreplication in
each treatment to minimize uncontrolled source of
variance like differences in the amount of powder
loaded in each well.
Data analyses
We estimated survival curves of cat flea of different
ages by the Kaplan–Meier method using the survfit
procedure of the library of survival of the statistical
package R. The survival curves of different ages
were compared by a log-rank test (survdiff procedure) in the same package. The effect of S. ctenocephali on the developmental time of C. felis were
evaluated for different oocyst concentrations by
using one-way analysis of variance (ANOVA) followed by the pairwise Tukey’s honest significance
difference (HSD) test. All analyses were performed
in the statistical environment R (R version 2·9·2;
Development Core Team).
RESULTS
Steinina ctenocephali is harmless to cat fleas
The effects of ingesting different concentrations
of gregarine oocysts on the emergence rates of
C. felis after three replicates are summarized in
Table 1. The survival of fleas exposed to S. ctenocephali
since the earliest larval stage, measured as emergence
rate of adult fleas was not altered by feeding on
different doses of gregarine oocysts spanning
0–50 oocysts mg−1. The adult emergence percentage
in exposed fleas ranged from 77 to 91%, a level that
falls within the range of previous reports (Hsu et al.
2002) and was essentially similar to the emergence
rate in the control group (87·5%) (Table 1). The
observed increase in dose 0 is noise but fall within
the ranges of the standard error estimate. Besides, no
significant difference was observed in survival rate of
flea larvae among replicates (P = 0·689, P = 0·168 and
P = 0·096, by log-rank test, respectively (Fig. 2).
This experiment demonstrates that overall survival
through development until adulthood is not affected
by gregarine infection, and it also shows that the two
feeding methods (i.e. SIP and artificial diet) are
optimum diets to reach adulthood.
Steinina ctenocephali accelerates the total development
time of the cat flea
In addition to measuring the effects of gregarine
infection on emergence rates, we also looked at the
duration of each stage to determine whether the
timing of development is affected. We reasoned
that by subtracting nutritional resources, the gregarine could alter, presumably delay the progression of
the larval stages. Our data show that the total
developmental time of fleas exposed to different
doses of gregarine oocysts did vary significantly
when compared to the control group (Table 2),
although in an unexpected way. Fleas fed on feces
containing gregarines developed much faster and
emerged earlier when compared to the control
group (P = 2·2 × 10−16, F = 24·62 for the first
instars larvae; P = 1·22 × 10−6, F = 8·84 for the
second instar larvae; P = 4·16 × 10−11, F = 15·38 for
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Gregarines effect on cat flea larvae development
5
Table 2. Effects of Steinina ctenocephali on the development of Ctenocephalides felis (±S.E. after three
replications).
Developmental time (days)
Oocysts dose
(oocysts mg−1)
L1
L2
L3
Pp
P
E–A
50
25
12·5
6·25
0·00
1·48 ± 0·05b
1·57 ± 0·05b
1·55 ± 0·04b
1·55 ± 0·04b
2·05 ± 0·05a
1·49 ± 0·07b
1·67 ± 0·05b
1·65 ± 0·06b
1·68 ± 0·06b
1·97 ± 0·04a
3·33 ± 0·08c
3·84 ± 0·15b
4·22 ± 0·12ab
4·17 ± 0·12ab
4·60 ± 0·13a
2·12 ± 0·08a
2·01 ± 0·07a
2·06 ± 0·08a
2·04 ± 0·06a
2·11 ± 0·07a
6·80 ± 0·17a
6·69 ± 0·13a
7·25 ± 0·20a
7·05 ± 0·21a
7·11 ± 0·17a
16·16 ± 0·24d
16·49 ± 0·24cd
17·76 ± 0·23b
17·40 ± 0·26bc
18·94 ± 0·30a
Means within a column followed by the same letter are not significantly different (Turkey’s HSD test, P < 0·05)
L1, first instar larvae; L2, second instar larvae; L3, third instar larvae; Pp, prepupa; P, pupa; E–A, eggs to adult.
the third instar larvae and P = 3·50 × 10−13, F = 18·97
for the emergence time, respectively) (Table 2). In
contrast, there is no difference in the developmental
time for prepupa and pupa between treated and
control groups (P = 0·7945, F = 0·42, prepupal stage
and P = 0·1384, F = 1·76 pupal stage, respectively).
DISCUSSION
Stray dog and cat populations in urban areas pose
critical sanitary, economic and social problems.
Ectoparasite infestation, which in turn leads to
spread of microbial pathogens, allergic conditions
and other health concerns, is one of the crucial problems associated with uncontrolled stray domestic
animals. Therefore, a deep understanding of the
biology of fleas is an important part of a comprehensive
effort to mitigate the problems mentioned above.
Fleas, like most insects are hosts to a number of microbial eukaryotes that establish symbiotic relationships
that are diverse in nature. Among them, gregarines
are ubiquitous, but only a few instances of gregarines
infecting fleas have been reported (Alarcón et al.
2011). A number of studies have addressed different
biological and ecological aspects of their interactions
(Beard et al. 1990; De Avelar and Linardi, 2008;
Alarcón et al. 2011). However, the effects of gregarine
infection on the flea, as well as the characteristics of
the symbiotic relationship between the partners it is
presently unknown. Gregarines are often considered
less harmful or even harmless to their hosts.
Nevertheless, they divert nutrients toward their own
growth, occupy space, and invoke immune reactions.
They also cause mechanical injury to gut epithelium
providing the port of entry for other microorganisms
into the body cavity of the host (Sneller, 1979); altering
longevity, and developmental time (Zuk, 2008).
Pathogenicity of gregarines has been mostly attributable to their trophozoite stage especially during attachment to host tissue and nutrient acquisition via
epimerite (Valigurová, 2012). In contrast, the effects
of trophozoites (Fig. 1C) derived from ingested sporozoites have received little attention. Cryptosporidium,
an apicomplexan parasite that is a close relative to
gregarine lineages (Templeton et al. 2010), is capable
of inducing apoptosis in infected cells by increasing
membrane permeability (McCole et al. 2000), which
ultimately results in cell death (Griffiths et al. 1994).
The apicomplexan invasion machinery comprehends
rhoptries, dense granules, and micronemes, which
are participating in recognition of and attachment to
the host cell, invasion, and formation of the parasitophorous vacuole where the sporozoite reside
(Dubremetz et al. 1998; Singh and Chitnis, 2012).
Gregarines exhibit similar ultrastructural features,
which along with similar modes of attaching to host
cells, and a robust phylogenetic association led to
propose that gregarine modes of infection could resemble early phases in the evolution of the obligate intracellular parasitism of apicomplexa (Leander, 2008). It is
thus possible that molecular determinants of pathogenesis and/or cell damage (e.g. cell death, gut epithelium injury) homologous to the apicomplexan invasion
machinery are also contributing to the extent of
damage caused by the gregarine sporozoite, in addition
to the energy expenditure from eliciting host defence.
The gregarine S. ctenocephali passes vertically
from flea’s parent to progeny via the fecal–oral
route, a mode of transmission that has been
confirmed by experimental infections with gregarine-infected feces from adult fleas (Beard et al.
1990; Alarcón et al. 2011). Sporozoites of S. ctenocephali are released into the gut of first instar larvae
from ingested infective oocysts, developing into
sporozoites that feed on the larval intestinal tissue.
The effect of different doses of oocysts on the survival and development of cat flea is presented
herein for the first time. The doses of S. ctenocephali-invading sporozoites tested are not affecting
adult emergence percentage or survival rate of
fleas. Our results demonstrate that the S. ctenocephali is not or least parasitic to the off-host stages
of the flea sharing symbiotic relationship (Table 1,
Suppl. figure 1). To our surprise, we found that
high doses of oocysts accelerated the total developmental time of C. felis. In other words, we observed
a significant reduction in the number of days spent in
the larval stages and the entire time length from eggs
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M. E. Alarcón and others
to adults when compared with the control group
(Table 2). From this and previous findings, we
suggest that S. ctenocephali has shaped a very intimate relationship with its host. This conclusion is
drawn by mainly two observations. First, Alarcón
et al. (2013) found that S. ctenocephali as well as C.
felis peaked simultaneously their populations in
summer under optimal conditions of temperature,
20–30 °C and humidity, 53–85% RH, and second,
the cycle of S. ctenocephali appears to be coordinated
with the host’s metamorphosis triggering their exit
from the host cells after the first blood meal of
newly emerged adult cat flea (Alarcón et al. 2011).
So, upon an adequate diet a healthy developing
larva transformed into adult without being lethally
affected by S. ctenocephali at this stage. Therefore,
the emergence rates of fleas were even higher than
that of previously reported in experimental setting
using uninfected fleas from our colony (Hsu and
Wu, 2000; Hsu et al. 2002). Altogether, our data
establish with a high degree of confidence that
S. ctenocephali is a well-adapted gregarine from the
cat flea. We have also unveiled a sophisticated mechanism of the gregarine/cat flea relationship in the
line of the manipulative hypothesis (Barnard and
Behnke, 1990; Poulin, 1994). Our interpretation is
that S. ctenocephali has developed the capacity of
accelerating, and therefore shortening the development time of the cat flea as a strategy to increase
their infection rate (fitness) and transmissibility
under favourable environmental conditions to flea
development. Noteworthy, L1, L2 and L3 larval
stages evidenced a clear alteration of their velocity
of development, whereas pre-pupa and pupa did
not, leading to speculate that at this point, the intracellular stage of the gregarine is predominantly
dormant. A high degree of specialization between
host and parasite is a well-known outcome of a long
history of coevolution, and it is strikingly illustrated
in a coordination of their life cycles (Rothschild and
Ford, 1964; Prensier et al. 2008). In some cases, the
arms race ensued at the establishment of a symbiotic
relationship results in the adoption of manipulative
strategies by the parasite. A frequent type of manipulation is the suppression of the immune response in
the host, which obviously facilitates the development
of the parasite’s stages associated with the host. In
other cases, the effect of the parasite can lead to
gross changes in morphology, behavioural alterations
(e.g. increased aggression), reproductive changes (e.g.
decreased mating, sterility), etc. Protozoa make only a
small fraction of the known cases of host manipulation. Moreover, the most thoroughly studied microbial manipulators are not strictly protozoa but fungi
(i.e. Microsporidia), which are known for some spectacular cases of feminizing and sex ratio distortion
effects on their hosts (Terry et al. 2004). A few examples of manipulators have been described from alveolates, the group of protists that includes gregarines,
6
apicomplexans and ciliates. For example, mosquitoes
infected with the ciliate Lambornella clarki Corliss
and Coats return to water and display a false oviposition behaviour, which helps the ciliate to infect more
mosquito larvae (Egerter et al. 1986). Some apicomplexan parasites are also known to manipulate their
hosts. Plasmodium species impair the blood clotting
ability of the mosquito, forcing it to take more but
smaller blood meals, resulting in more infected
hosts (Koella et al. 2002), whereas Toxoplasma can
induce elevated aggression and reduces defensive
behaviour in mice and rats, increasing the exposure
to predators (e.g. cats), which helps the parasite to
spread (Webster and Brunton, 1994; Lagrue and
Poulin, 2010). Finally, intracellular stages of gregarines’ closest relatives Cryptosporidium parvum
deploy strategies to modulate the apoptotic programme of the host cell to their favour (Heussler
et al. 2001).
This study shows that the gregarine S. ctenocephali
manipulates its host, the cat flea, inducing a notable
acceleration of the larval stages. The signal cues
behind this acceleration of cat flea development are
unknown, but may be hormonal in nature, perhaps
triggered by the first blood meal of newly emerged
adults (Alarcón et al. 2011). Interestingly, early
researchers had noticed hormone-associated correlations between both protozoan flagellates and particular
gregarines (Cleveland, 1959; Corbel, 1964; PorchetHenneré, 1969). Indeed, gregarines infecting flies of
genus Schneideria Kochetkova are examples of very
particular and unique concomitance host’s brain hormones and parasites (Nowlin, 1922; Malavasi, 1976).
Identifying the molecular basis of the symbiontmediated manipulation of the cat flea is very important to understand the significance and evolution of
this symbiotic relationship in the context of the
origins of parasitism in Apicomplexa. In addition,
this avenue of research can lead to artificial disruption
of the cat flea development, and contribute to spawn a
new generation of biological control strategies.
SUPPLEMENTARY MATERIAL
The supplementary material for this article can be
found at https://doi.org/10.1017/S0031182016002122
ACKNOWLEDGMENTS
We sincerely thank W. J. Wu (NTU) for providing logistic
support, suggestions and laboratory facilities; the staffs of
Animal Shelter, Taipei (Taiwan) for access to flea sampling;
to Y. C. Hsu, M. L. Daza for assisting in flea collections; to
M. SM. Margarita for facilities in the Universidad de
Concepción, Chile, and to CHS, FCIAR for partly facilitating this research by a grant (no. 386345) from the Natural
Sciences and Engineering Research Council, Canada.
This study complies with the current law of Taiwan
(ROC). This work was carried out at the Division of
Entomology, National Taiwan University, 1st Roosevelt
Road, Taipei, Taiwan (Republic of China).
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Gregarines effect on cat flea larvae development
FINANCIAL SUPPORT
This research was supported by National Science Council,
Taiwan under graduate fellowship (no. 96-2313-B002053-MY3) to the first author.
AUTHOR CONTRIBUTIONS
M. E. A. conceived, designed and performed the
experiments; M. E. A., A. K. D., A. J.-F., R. E. B.
and C. H. S. analysed the data; M. E. A., A. J.-F.,
R. E. B. and C. H. S. contributed reagents/materials/
analysis tools; M. E. A., A. K. D., A. J.-F. and
C. H. S. wrote the paper.
REFERENCES
Alarcón, M. E., Huang, C. G., Tsai, Y. S., Chen, W. J., Dubey, A. K.
and Wu, W. J. (2011). Life cycle and morphology of Steinina ctenocephali
(Ross 1909) comb. nov. (Eugregarinorida: Actinocephalidae), a gregarine
of Ctenocephalides felis (Siphonaptera: Pulicidae) in Taiwan. Zoological
Studies 50, 763–772.
Alarcón, M. E., Huang, C. G., Dubey, A. K. and Benitez, H. A. (2013).
A gregarine from the gut of cat flea, Ctenocephalides felis (Bouche)
(Siphonaptera: Pulicidae) in Taiwan: dynamic of infection patterns.
Veterinary Parasitology 192, 51–56.
Barnard, C. J. and Behnke, J. M. (1990). Parasitism and Host Behaviour.
Taylor and Francis Publisher, London, UK.
Beard, C. B., Butler, J. F. and Hall, D. W. (1990). Prevalence and
biology of endosymbionts of fleas (Siphonaptera: Pulicidae) from dogs
and cats in Alachua County, Florida. Journal of Medical Entomology 27,
1050–1061.
Bossard, R. L., Dryden, M. W. and Broce, A. B. (2002). Insecticide susceptibilities of cat fleas (Siphonaptera : Pulicidae) from several regions of
the United States. Journal of Medical Entomology 39, 742–746.
Cleveland, L. R. (1959). Sex induced with ecdysone. Proceedings of the
National Academy of Sciences of the United States of America 45,
747–753.
Corbel, J. C. (1964). Infestations expérimentales de Locusta migratoria
L. (insecte orthoptère) par Gregarina garnhami Canning (Sporozoaire gregarinomorphe): relation entre le cycle de l’hôte et celui du parasite. Comptes
Rendus de l’Académie des Sciences. Biologies 259, 207–210.
De Avelar, D. M. and Linardi, P. M. (2008). Seasonality and prevalence
rates of Steinina sp. (Eugregarinorida: Actinocephalidae) in Ctenocephalides
felis felis (Siphonaptera: Pulicidae) from dogs captured in Belo Horizonte,
Minas Gerais, Brazil. Journal of Medical Entomology 45, 1139–1142.
Dubremetz, J. F., Garcia-Reguet, N., Conseil, V. and Fourmaux, M.
N. (1998). Apical organelles and host-cell invasion by Apicomplexa.
International Journal for Parasitology 28, 1007–1013.
Egerter, D. E., Anderson, J. R. and Washburn, J. O. (1986). Dispersal of
the parasitic ciliate Lambornella clarki: implications for ciliates in the biological control of mosquitoes. Proceedings of the National Academy of
Sciences of the United States of America 83, 7335–7339.
Griffiths, J. K., Moore, R., Dooley, S., Keusch, G. T. and Tzipori, S.
(1994). Cryptosporidium parvum infection of Caco-2 cell monolayers
induces an apical monolayer defect, selectively increases transmonolayer permeability, and causes epithelial cell death. Infection and Immunity 62, 4506–
4514.
Henderson, G., Manweiler, S. A., Lawrence, W. J., Tempelman, R.
J. and Foil, L. D. (1995). The effects of Steinernema carpocapsae (Weiser)
application to different life stages on adult emergence of the cat flea
Ctenocephalides felis (Bouche). Veterinary Dermatology 6, 159–163.
Heussler, V. T., Kuenzi, P. and Rottenberg, S. (2001). Inhibition of
apoptosis by intracellular protozoan parasites. International Journal for
Parasitology 31, 1166–1176.
Hsu, Y. C. (2000). Effects of diet factors on larval development and survival
of cat fleas, Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae).
Master thesis. National Taiwan University, Taipei (in Chinese, English
[abstract]), 101 pp.
Hsu, M. H. and Wu, W. J. (2000). Effects of multiple mating on female
reproductive output in the cat flea (Siphonaptera: Pulicidae). Journal of
Medical Entomology 37, 828–834.
7
Hsu, M. H., Hsu, Y. C. and Wu, W. J. (2002). Consumption of flea faeces
and eggs by larvae of the cat flea, Ctenocephalides felis. Medical and
Veterinary Entomology 16, 445–447.
Koella, J. C., Rieu, L. and Paul, R. (2002). Stage-specific manipulation
of a mosquito’s host-seeking behaviour by the malaria parasite
Plasmodium gallinaceum. Behav. Behavioural Ecology 13, 816–820.
Lagrue, C. and Poulin, R. (2010). Manipulative parasites in the world of
veterinary science: implications for epidemiology and pathology.
Veterinary Journal 184, 9–13.
Leander, B. S. (2008). Marine gregarines: evolutionary prelude to the apicomplexan radiation? Trends in Parasitology 24, 60–67.
Malavasi, A. (1976). Relationships between the gregarine Schneideria
schneiderae and its host Trichosia pubescens (Diptera, Sciaridae). Journal
of Invertebrate Pathology 28, 363–371.
McCole, D. F., Eckmann, L., Laurent, F. and Kagnoff, M. F. (2000).
Intestinal epithelial cell apoptosis following Cryptosporidium parvum infection. Infection and Immunity 68, 1710–1713.
McCoy, C., Broce, A. B. and Dryden, M. W. (2008). Flea blood feeding
patterns in cats treated with oral nitenpyram and the topical insecticides
imidacloprid, fipronil and selamectin. Veterinary Parasitology 156,
293–301. Epub 2008 May 23.
Nowlin, N. (1922). Correlation of the life cycle of a parasite with the metamorphosis of its host. Journal of Parasitology 8, 153–160.
Porchet-Henneré, E. (1969). Corrélations entre le cycle d’une Coccidie:
Coelotropha durchoni Vivier, et celui de son hôte Nereis diversicolor
O. F. Müller (Annélide Polychète). Zeitschrift für Parasitenkunde 31,
299–314.
Poulin, R. (1994). The evolution of parasite manipulation of host behaviour: a theoretical analysis. Parasitology 109, 109–118.
Prensier, G., Dubremetz, J. F. and Schrevel, J. (2008). The unique
adaptation of the life cycle of the coelomic gregarine Diplauxis hatti to its
host Perinereis cultrifera (Annelida, Polychaeta): an experimental and ultrastructural study. Journal of Eukaryotic Microbiology 55, 541–553.
Ross, E. (1909). A gregarine parasitic in the dog-flea Ctenocephalus serraticeps. Annals of Tropical Medicine and Parasitology 2, 359–363.
Rothschild, M. and Ford, B. (1964). Maturation and egg-laying of the
rabbit flea (Spilopsyllus cuniculi Dale) induced by the external application
of hydrocortisone. Nature 203, 210–211.
Rueckert, S., Chantangsi, C. and Leander, B. S. (2010). Molecular systematics of marine gregarines (Apicomplexa) from North-eastern Pacific
polychaetes and nemerteans, with descriptions of three novel species:
Lecudina phyllochaetopteri sp. nov., Difficilina tubulani sp. nov. and
Difficilina paranemertis sp. nov. International Journal of Systematic and
Evolutionary Microbiology 60, 2681–2690.
Rust, M. K., Hinkle, N. C., Waggoner, M., Mencke, N., Hansen, O.
and Vaughn, M. B. (2001). The influence of imidacloprid on adult cat
flea feeding. Compendium on Continuing Education for the Practicing
Veterinarian 23, 18–21.
Singh, S. and Chitnis, C. E. (2012). Signalling mechanisms involved in
apical organelle discharge during host cell invasion by apicomplexan parasites. Microbes and Infection/Institut Pasteur 14, 820–824.
Sneller, V. P. (1979). Inhibition of Dirofilaria immitis in gregarineinfected Aedes aegypti preliminary observations. Journal of Invertebrate
Pathology 34, 62–70.
Templeton, T. J., Enomoto, S., Chen, W. J., Huang, C. G., Lancto, C.
A., Abrahamsen, M. S. and Zhu, G. (2010). A genome-sequence survey
for Ascogregarina taiwanensis supports evolutionary affiliation but metabolic diversity between a Gregarine and Cryptosporidium. Molecular
Biology and Evolution 27, 235–248.
Terry, R. S., Smith, J. E., Sharpe, R. G., Rigaud, T., Littlewood, D. T.,
Ironside, J. E., Rollinson, D., Bouchon, D., MacNeil, C., Dick, J. T. A.
and Dunn, A. M. (2004). Widespread vertical transmission and associated
host sex-ratio distortion within the eukaryotic phylum Microspora. Proceedings
of the Royal Society of London Series B, Biological Sciences 271, 1783–1789.
Thomas, R. E., Wallenfels, L. and Popiel, I. (1996). On-host viability and
fecundity of Ctenocephalides felis (Siphonaptera: Pulicidae), using a novel
chambered flea technique. Journal of Medical Entomology 33, 250–256.
Valigurová, A. (2012). Sophisticated adaptations of Gregarina cuneata
(Apicomplexa) feeding stages for epicellular parasitism. PLoS ONE 7,
e42606. Epub 2012 August 10.
Webster, J. P. and Brunton, C. F. A. (1994). Macdonald DW. Effect of
Toxoplasma gondii upon neophobic behaviour in wild brown-rats,
Rattusm norvegicus. Parasitology 109, 37–43.
Zuk, M. (2008). The effects of gregarine parasites on longevity, weight loss,
fecundity and developmental time in the field crickets Gryllus veletis and G.
pennsylvanicus. Ecological Entomology 12, 349–354.
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