# Gregarine parasites are adapted to mosquito winter diapause

Edwige Martin, Laurent Vallon, Camille da Silva Carvalho, Maxime Girard, Guillaume Minard

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Edwige Martin, Laurent Vallon, Camille da Silva Carvalho, Maxime Girard, Guillaume Minard. Gregarine parasites are adapted to mosquito winter diapause. Parasites & Vectors, 2022, 15 (1), pp.249. 10.1186/s13071-022-05365-w. https://hal.inrae.fr/hal-03788131

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# Gregarine parasites are adapted to mosquito winter diapause

Edwige Martin†, Laurent Vallon†, Camille Da Silva Carvalho, Maxime Girard and Guillaume Minard*

**Abstract**

The Asian tiger mosquito *Aedes albopictus* is one of the most invasive species of mosquito. The prevalence of its apicomplexan gregarine parasite *Ascogregarina taiwanensis* is high in natural populations across both temperate and tropical regions. However, the parasite's oocysts cannot colonize the insect host during winter, when the mosquito lays diapausing eggs. It is therefore unclear if the parasite can survive outside of its insect host during the cold season in temperate regions. Oocysts stored for 1 month at a low temperature (representative of the temperatures that occur during periods of mosquito diapause) were as infectious as fresh oocysts, but those stored for the same period of time at a higher temperature (representative of the temperatures that occur during periods of mosquito activity) were uninfectious. We therefore suggest that the parasite has evolved traits that maximize its maintenance during periods of mosquito dormancy, while traits that would enable its long term survival during periods of mosquito activity have not been selected for.

**Keywords:** *Ascogregarina*, *Aedes albopictus*, Thermal tolerance, Apicomplexa

The phylum Apicomplexa, which comprises heterotrophic animal parasites, evolved from photosynthetic marine algae [1]. The Apicomplexa have been intensively studied because important human pathogens belong to this phylum, such as *Toxoplasma* and *Plasmodium*. Gregarine parasites (Gregarinasina) are of great interest from an evolutionary perspective since their lineage is the most ancient of the Apicomplexa. They are highly diverse and infect a large number of invertebrates across marine, freshwater and terrestrial ecosystems [2]. Their interactions with their hosts are complex, and range from mutualism to parasitism.

In mosquitoes, only one gregarine genus has been reported, namely *Ascogregarina* [3]. Species of the genus *Ascogregarina* mainly colonize *Aedes* spp., but also colonize *Tripteroides dofleini* and *Armigeres subalbatus*, and

show high host specificity. They are often considered weakly pathogenic to their natural hosts but harmful to other species [4]. The Asian tiger mosquito *Aedes albopictus* is one of the most invasive species of mosquitoes in the world [5]. Originating in Asia, it has efficiently colonized every continent, except for Antarctica, over the past century [6]. Its ability to lay diapausing eggs which can remain dormant during the winter has enabled it to survive low temperatures and colonize temperate regions [7]. Its gregarine parasite *Ascogregarina taiwanensis* has a remarkable prevalence in both native and introduced populations [8–11], although some exceptions have been reported [12]. The parasites are released as oocysts (a means of environmental resistance) by adult mosquitoes when these emerge, die or defecate in freshwater [3]. The oocysts infect 1st instar mosquito larvae through ingestion in their freshwater habitat, and the biological cycle of the parasite, which occurs within the digestive tract of the insect (i.e. the gut lumen, intestinal epithelium and lumen of the Malpighian tubules) is regulated by the development of the mosquito [13, 14]. It is, however, unclear whether the parasite can remain infectious

<footnote>†Edwige Martin and Laurent Vallon contributed equally to this work</footnote>

<footnote>*Correspondence: guillaume.minard@univ-lyon1.fr</footnote>

<footnote>UMR Ecologie Microbienne, Université Claude Bernard Lyon 1, CNRS, INRAe, VetAgro Sup, 69622 Villeurbanne, France</footnote>
during the winter period. In this study, we aimed to test
the infective ability of oocysts under winter and summer
conditions.

We generated a line of mosquitoes from a focal popu-
lation collected in September 2017 in Villeurbanne and
Pierre-Bénite (France). The mosquitoes were reared
under laboratory conditions at 28 °C. The presence of A.
taiwanensis in the line was confirmed by diagnostic poly-
merase chain reaction following the protocol described
by Reyes-Villanueva et al. [15], and by microscopic
observation of the gut in pupae and Malpighian tubules
in adults. The parasite was maintained in the line over
22 generations by (i) sprinkling the crushed bodies of
infected adults over the water in the containers holding
the larvae, (ii) not storing eggs, and (iii) never changing
the water in the containers before the 2nd instar stage. A
non-infected line was generated from the same focal pop-
ulation of mosquitoes and maintained for 18 generations.
This was achieved by (i) treating F₁ mosquito eggs with
griseofulvin (1 mg ml⁻¹), a fungicide that shows antipara-
sitic activity against gregarines [16]; and (ii) changing the
water in which the 1st instar larvae were maintained. We
verified the absence of the parasite by microscopy.

To test our hypothesis, we exposed 100 uninfected
mosquito larvae to fresh oocysts or oocysts stored for
1 month at a low temperature (4 °C), or oocysts stored
for 1 month at a higher temperature (26 °C). The oocysts
were obtained by crushing 100 infected adult mosquitoes
in 45-ml sterile water in a Dounce tissue grinder tube
(mean ± SD = 2.56 ± 2.14 × 10⁶ oocysts). The contents of
each tube were then separated into three 15-ml tubes.
The contents of one of these tubes was used to imme-
diately re-infect mosquitoes, while the contents of the
other tubes were stored at 4 °C or 26 °C. The experiment
was replicated for three different mosquito batches per
condition. A negative control consisting of uninfected
mosquitoes was prepared to ensure that there was no
oocyst material in the test population. We then counted
the number of oocysts that had colonized the adult
individuals that emerged under each of the test condi-
tions. Survival to immature stages was slightly higher
for uninfected individuals (mean ± SD = 87.67 ± 3.29%),
individuals colonized with parasites storedat 26 °C
(mean ± SD = 93.67 ± 0.58%) and individuals immediately
reinfected with parasites (mean ± SD = 85.67 ± 5.03%)
compared to those reinfected with parasites stored at
4 °C (mean ± SD = 75.33 ± 3.06%).

Individual mosquitoes were crushed with a ster-
ile pestle in 100 µl of sterile water (Gibco, France).
Oocysts were counted in 10 haemocytometer cells
per mosquito at ×400 magnification under a micro-
scope (Leica, Germany). Number of oocysts per

mosquito was calculated using the following equation:
n(oocysts/mosquito) = Water volume × Average number of oocyst per cell / Cell volume
Oocyst counts were determined for a total of 10 males and
10 females per condition and per replicate (i.e. a total of
240 individuals).

Statistical analyses were performed with R software v. 4.0.2 (R Development Core Team). The data were plotted using the ggplot2 and ggpubr R packages.

Interestingly, no oocysts were retrieved from the major-
ity of mosquitoes infected with oocysts stored at 26 °C
(Fig. 1), or from the uninfected individuals. These results
differed from those for mosquitoes infected with fresh
oocysts or oocysts stored at 4 °C. These observations were
consistent across sexes and replicate batches. Since the
data were zero-inflated, the oocyst count data were trans-
formed into presence–absence for the analysis, where
presence indicated that an individual was parasitized and
absence indicated that it was not. Variation in the oocyst
infection rate between replicate batches, male and female
mosquitoes, and experimental conditions were modelled
with a generalized linear model with a binomial distribu-
tion using the brglm2 R package. The influence of each
explanatory variable on the response variables was tested
by type II Wald χ² with the car R package. This showed
that oocyst infection rate was significantly influenced by
the experimental condition (Wald χ², P < 2 × 10⁻¹⁶) and
by the sex (Wald χ², P = 0.02) of the mosquito in interac-
tion with the replicate batch (Wald χ², P = 0.02; Table 1).
Therefore, independent analyses were repeated for each
sex separately. Oocyst infection rate variation among
mosquitoes of both sexes was significantly affected by
the experimental conditions tested (Wald χ², females,
P < 2 × 10⁻¹⁶; males, P = 7.98 × 10⁻¹⁵), and differences
were also observed between batches for males (Wald χ²,
P = 3.78 × 10⁻⁴; Table 1). After averaging the values of the
batch replicates, post hoc comparisons of pairwise experi-
mental conditions were performed using Tukey hon-
est significant difference (HSD) test with the emmeans
R package. These pairwise comparisons indicated that
females and males showed consistent patterns despite the
batch effect observed for the males (Table 2). The pairwise
comparisons showed that oocytes stored in water under
a low temperature were still infectious, and that infection
rates under this condition did not significantly differ from
those in which fresh oocysts were used to infect mosqu-
toes (Tukey HSD, females, P = 0.3; males, P = 0.98). In
contrast, rates of infection with oocysts stored in 26 °C
water were consistently lower than those with fresh
oocysts or oocysts stored at 4 °C (Tukey HSD, P < 10⁻²
for each comparison across both sexes), and did not signifi-
cantly differ from those of uninfected control individuals
(Tukey HSD, males, P = 0.99; females, P = 0.99).
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Number of oocysts per adult mosquito across the experimental conditions. Females (blue) and males (yellow) were tested across three independent replicate batches. The violin plots show the log₁₀ of total oocyst number + 1 across the experimental conditions. In the Fresh oocysts treatment, infected mosquitoes were crushed into sterile water which was immediately used to seed the water in the tray containing the unparasitized 1st instar larvae. Non-infected mosquitoes were unparasitized and used as a control to confirm that no residual parasites had colonized the test population. In the Parasite kept 1 month at 26 °C or 4 °C treatments, infected mosquitoes were crushed in sterile water and stored for 1 month at 26 °C or at 4 °C, respectively before being seeded to tray containing unparasitized 1st instar larvae. Larvae were reared to adult mosquitoes before parasitism measurement</figcaption></figure>

<table><caption>**Table 1** Factors explaining variation in the oocyst infection rate</caption><thead><tr><th>Dataset</th><th>Response variable</th><th>Explanatory variable</th><th>Wald χ²</th><th>df</th><th>P-value</th><th>Significance</th></tr></thead><tbody><tr><td rowspan="7">Full</td><td rowspan="7">Oocyst infection (presence/absence)</td><td>Condition</td><td>142.668</td><td>3</td><td>&lt;2 × 10<sup>-16</sup></td><td>***</td></tr><tr><td>Batch</td><td>7.719</td><td>2</td><td>0.02</td><td>*</td></tr><tr><td>Sex</td><td>2.438</td><td>1</td><td>0.12</td><td></td></tr><tr><td>Condition × Batch</td><td>1.960</td><td>6</td><td>0.92</td><td></td></tr><tr><td>Condition × Sex</td><td>1.254</td><td>3</td><td>0.74</td><td></td></tr><tr><td>Batch × Sex</td><td>8.110</td><td>2</td><td>0.02</td><td>*</td></tr><tr><td>Condition × Batch × Sex</td><td>-9.087</td><td>6</td><td>1</td><td></td></tr><tr><td rowspan="3">Females</td><td rowspan="3">Oocyst infection (presence/absence)</td><td>Condition</td><td>76.508</td><td>3</td><td>&lt;2 × 10<sup>-16</sup></td><td>***</td></tr><tr><td>Batch</td><td>1.849</td><td>2</td><td>0.4</td><td></td></tr><tr><td>Condition × Batch</td><td>-2.976</td><td>6</td><td>1</td><td></td></tr><tr><td rowspan="3">Males</td><td rowspan="3">Oocyst infection (presence/absence)</td><td>Condition</td><td>68.730</td><td>3</td><td>7.98 × 10<sup>-15</sup></td><td>***</td></tr><tr><td>Batch</td><td>15.764</td><td>2</td><td>3.78 × 10<sup>-4</sup></td><td>***</td></tr><tr><td>Condition × Batch</td><td>-4.150</td><td>6</td><td>1</td><td></td></tr></tbody></table>

<footnote>* P < 0.05, ** P < 0.01, *** P < 0.001</footnote>
<table><caption>**Table 2** Pairwise comparisons of oocyst infection rate between conditions</caption><thead><tr><th>Dataset</th><th>Condition 1 in the comparison</th><th>Condition 2 in the comparison</th><th>z-ratio</th><th>P-value</th><th>Significance</th></tr></thead><tbody><tr><td rowspan="6">Females</td><td>Parasite stored for 1 month at 26 °C</td><td>Parasite stored for 1 month at 4 °C</td><td>-3.353</td><td>4.4 × 10<sup>-3</sup></td><td>**</td></tr><tr><td>Parasite stored for 1 month at 26 °C</td><td>Infection with fresh oocysts</td><td>-4.344</td><td>1 × 10<sup>-4</sup></td><td>***</td></tr><tr><td>Parasite stored for 1 month at 26 °C</td><td>Non-infected mosquitoes</td><td>0.341</td><td>0.99</td><td></td></tr><tr><td>Parasite stored for 1 month at 4 °C</td><td>Infection with fresh oocysts</td><td>-1.757</td><td>0.30</td><td></td></tr><tr><td>Parasite stored for 1 month at 4 °C</td><td>Non-infected mosquitoes</td><td>3.459</td><td>3 × 10<sup>-3</sup></td><td>**</td></tr><tr><td>Parasite stored for 1 month at 4 °C</td><td>Infection with fresh oocysts</td><td>4.372</td><td>1 × 10<sup>-4</sup></td><td>***</td></tr><tr><td rowspan="6">Males</td><td>Parasite stored for 1 month at 26 °C</td><td>Parasite stored for 1 month at 4 °C</td><td>-3.351</td><td>4.5 × 10<sup>-3</sup></td><td>**</td></tr><tr><td>Parasite stored for 1 month at 26 °C</td><td>Infection with fresh oocysts</td><td>-3.601</td><td>1.8 × 10<sup>-3</sup></td><td>**</td></tr><tr><td>Parasite stored for 1 month at 26 °C</td><td>Non-infected mosquitoes</td><td>0</td><td>1</td><td></td></tr><tr><td>Parasite stored for 1 month at 4 °C</td><td>Infection with fresh oocysts</td><td>-0.396</td><td>0.98</td><td></td></tr><tr><td>Parasite stored for 1 month at 4 °C</td><td>Non-infected mosquitoes</td><td>3.351</td><td>4.5 × 10<sup>-3</sup></td><td>**</td></tr><tr><td>Parasite stored for 1 month at 4 °C</td><td>Infection with fresh oocysts</td><td>3.601</td><td>1.8 × 10<sup>-3</sup></td><td>**</td></tr></tbody></table>

In temperate regions, the active period of mosquitoes ranges from April/May to October/November, while during the rest of the year mosquitoes lay diapausing eggs that remain dormant until the following active period [17]. *Ascogregarina taiwanensis* colonizes every developmental stage of mosquitoes except for the egg stage [13]. Therefore, it cannot infect its mosquito hosts during the period of dormancy, and thus persists as a free-living oocyst until mosquito eggs hatch. However, one can hypothesize that the parasite may infect its host during the inactive period if life stages other than eggs are maintained during the winter. Adult hibernation has been reported in some mosquito species [18] but not, to the best of our knowledge, in *Ae. albopictus*. Mosquitoes can pursue their cycle over the winter if they find warm refuges. However, *Ae. albopictus* mostly colonizes open habitats [19]. The overwintering of mosquitoes in greenhouses have been reported in the Netherlands, but this remains a solitary example of this behaviour [20]. Concrete tanks were used as winter refuges for larvae and pupae in northern Vietnam [21], but the winter temperatures in this subtropical region average 14 °C, which is sufficiently high to allow the slow but complete development of the mosquito. In contrast, open bodies of water in temperate regions can reach lower temperatures in winter.

The prevalence of *Ascogregarina taiwanensis* in *Aedes albopictus* can reach 100% in temperate regions [11]. This finding is inconsistent with low survival of the parasite in mosquito refuges during the winter. However, based on our results, we can propose an explanation for this discrepancy. Since the oocysts showed no decrease in infectivity when they were stored in water at a low temperature (at least 1 month at 4 °C), we suggest that the parasite is adapted to survive outside of its host during the latter's period of inactivity. It is interesting

to note that the survival of the parasite at the higher temperature tested, which is representative of temperatures that occur during the period of mosquito activity, was poor. A possible explanation for this is that the parasite is more likely to encounter a mosquito host in warmer periods, and thus there may be less selective pressure for resistance in oocysts at temperatures prevalent during the mosquito's active season.

In conclusion, our study provides an example of parallel phenology which may explain the success of a neglected, but widespread, invasive parasitic species in infecting its mosquito host. This profile resembles that of *Cryptosporidium*, an apicomplexan parasite of mammals that is transmitted in water. In that model, infectivity remained stable for at least 12 weeks when the oocysts were stored in water at 4 °C, but dropped dramatically after 6 weeks when the oocysts were stored at 25 °C [22]. This type of pattern could have epidemiological consequences, since transmission rates of viruses and filarial nematodes in *Aedes* mosquitoes have been shown to vary according to the degree to which the mosquitoes have been parasitized by *Ascogregarina* spp. [23, 24].

### Acknowledgements

We thank the Master's in Microbiology Programme and the Ecology, Evolution, Microbiology and Modelling Doctoral School of the University of Lyon, to which both CDC and MG are associated.

### Author contributions

EM, LV and GM designed the experiments. EM and LV conducted the experiments with the help of CDC, MG and GM. GM performed the statistical analysis. GM wrote the first draft of the manuscript and all the co-authors reviewed it. All the authors read and approved the final manuscript.

### Funding

This study was funded by the EC2CO Project (grant INTERASCO) and the ANSES Project (grant AS-CONTROL PNR EST 2019-1-095).

<footnote>* P < 0.05, ** P < 0.01, *** P < 0.001</footnote>
**Availability of data and materials**

The datasets supporting the conclusions of this article are included within the article and in its additional file.

**Declarations**

**Ethics approval and consent to participate**

Experiment on insects are exempted from ethics approval. To produce the mosquito eggs, the adult females were fed on anaesthetized mice (*Mus musculus*). This protocol was approved by an ethical committee and validated by the French Ministry for Higher Education and Research (APAFIS#31807-2021052715018315 v2).

**Consent for publication**

Not applicable.

**Competing interests**

The authors declare that they have no competing interests.

Received: 26 March 2022 Accepted: 12 June 2022

Published online: 11 July 2022

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