Código ISSN 2588-0551

https://www.inspilip.gob.ec

Magazine INSPILIP - V 6 - Number 1 - May 2022

Carrazco A , Ponce P, Lagos

P, Coloma J, Varsovia C.

Parasitism of Ascogregarina

culicis (Apicomplexa:

Lecudinidae) in immature stages

and adults of Aedes aegypti

(Diptera: Culicidae) from

Ecuador.  

INSPILIP. 2022; 6 (1).

The author declares to be free of any

personal or commercial association

     

interest in connection with the article,

as well as having respected the

ethical principles of research, such as

   

of the institution where the study

was carried out. , permission to use

the data, informed consent and in

the case of observational studies and

   

a CEISH, ARCSA, Environment,

among others, according to the

category. In addition, the license

to publish images of the person or

persons that appear in the manuscript.

For this reason, INSPILIP is not

    

third parties, neither is INSPI as the

publishing entity, nor the Editor, the

responsibility of the publication is

the absolute responsibility of the

authors.





Patricio Ponce Ph.D.











Varsovia Cevallos, Ph.D.



a. National Institute of Public Health Research; National Reference Center for Genomics,

Sequencing and Bioinformatics (GENSBIO), Quito 170136, Ecuador



7360.

Correspondence: Varsovia Cevallos, Ph.D. Email:  



Identication of the responsibility and contribution of the authors:

          

   

         



P.P., and J.C.; project administration, V.C., P.P, and J.C.; funding acquisition, V.C. and P.P. All

authors have read and agreed to the published version of the manuscript.

Date of admission:  Approval date:  Publication date: 

Parasitism of Ascogregarina culicis (Apicomplexa: Lecudinidae) in immature

stages and adults of Aedes aegypti (Diptera: Culicidae) from Ecuador.

Parasitismo de Ascogregarina culicis (Apicomplexa: Lecudinidae) en estadios

inmaduros y adultos de Aedes aegypti (Diptera: Culicidae) de Ecuador.

DOI: https://doi.org/10.31790/inspilip.v6i1.267

Original article

Open access

Abstract

Citatión

Objectives: Aedes aegypti and Ae. albopictus transmit several medically important



that invade the midgut epithelial cells, where they complete their life cycles. There

are no reports of the presence of this parasite associated with Aedes species in

Ecuador. This study aimed to identify the presence of

Ascogregarina in naturally

infected Ae. aegypti and Ae. albopictus that were collected in several localities of

       

by microscopy and by sequencing using a ssrRNA conserved region. Results: We

      

Ascogregarina culicis infecting Aedes

aegypti from Ecuador. The parasite was found in 30 adult and immature mosquitoes



and highlights the importance of studying the parasitism of mosquitoes for biological

control and their possible role in arbovirus transmission.

Keywords: Parasites. Culicidae. Aedes. Apicomplexa. Entomology.

Código ISSN 2588-0551

https://www.inspilip.gob.ec

Parasitism of Ascogregarina culicis (Apicomplexa: Lecudinidae) in immature stages and adults of Aedes aegypti

Carrazco Montalvo A

Magazine INSPILIP - V 6 - Number 1 - May 2022

Resumen

Objetivos: Aedes aegypti    Ae.

albopictus 

de importancia médica a los seres humanos. Estos

    

gregarinos que invaden las células epiteliales del

intestino medio, donde completan sus ciclos de

vida. No existen reportes de la presencia de este

parásito asociado a especies del género Aedes en



la presencia de Ascogregarina en Ae. aegypti y Ae.

albopictus recolectados en varias localidades del

    Ascogregarina fue

     

     

Resultados: Presentamos el primer registro

  Ascogregarina culicis infectando

naturalmente Aedes aegypti en Ecuador. El parásito





      

destaca la importancia de estudiar el parasitismo de la

microbiota de los mosquitos, y su uso potencial en el



del arbovirus a los humanos.

Palabras clave: Parásitos. Culicidae. Aedes.



Introduction

Aedes aegypti    Ae. albopictus



      

     

others

     

parasites of the genus Ascogregarina (Apicomplexa:

Lecudinidae). Ascogregarina culicis  

    Ae. aegypti



in urban and

suburban habitats

and Ascogregarina taiwanensis

have been recorded in Ae. albopictus.

Aedes aegypti is widely distributed in tropical and

subtropical areas of continental Ecuador and the

Galápagos Islands, while Aedes albopictus 

      



The parasite Ascogregarina oocysts are ingested by

       

infecting the gut epithelial cells, and progressing

       

    

    

      

sexual gametes

The gametocyte contains hundreds of oocysts during

the pupal stage and is released into the environment

after the metamorphosis of pupae into adults. The

transmission of these parasites to other mosquitoes

occurs when oocysts released by defecation of



The life cycle of the parasites seems to be completely

adapted to the mosquito life cycle, suggesting strong





Ascogregarina taiwanensis

and Ascogregarina culicis has been reported in larvae

of Ae. albopictus and Ae. aegypti, respectively, with

similar mean infection intensities in both species of

mosquito



The role of the midgut microbiota of Ae. aegypti may

contribute to understanding its population dynamics

and its vectorial capacity of viral diseases



There are no reports of the presence of this parasite

associated with Aedes species in Ecuador. This study

aimed to identify the presence of Ascogregarina in

naturally infected Ae. aegypti and Ae. albopictus that

were collected in several localities of Ecuador.

Materials and Methods

Collection of specimens

Immature individuals were collected in plastic

containers and transported to the laboratory for adult



aspirator and stored individually in 1.5 ml tubes. The

geographical coordinates of each collection location

were recorded.

Immature stages and adults of Aedes aegypti and

Aedes albopictus were collected in urban and rural

     

areas in Ecuador (Table 1).

   Ae. aegypti were collected in

    



Domingo de los Tsáchilas (Andean lowlands); Tena,

Nueva Loja (Lago Agrio), Francisco de Orellana,

    

Código ISSN 2588-0551

Parasitism of Ascogregarina culicis (Apicomplexa: Lecudinidae) in immature stages and adults of Aedes aegypti

Carrazco Montalvo A

Código ISSN 2588-0551

https://www.inspilip.gob.ec

Magazine INSPILIP - V 6 - Number 1 - May 2022

Voucher samples were deposited at the National

Collection of Arthropod Vectors of the “Instituto





        

adults of Ae. aegypti    

the same number of samples of Ae. albopictus in



Aedes aegyp�

egion

rovince

ocality

a�tude

Longitude

Collec�on

ear

Number of

pecimens

aciﬁc coast

smeraldas

.9592

79.6539

014

anabí

anta

0.9621

80.7127

014

uayas

uayaquil

2.1961

79.8862

014

os Ríos

abahoyo

1.8021

79.5344

014

anabí

hone

0.6981

80.0936

014

anabí

ortoviejo

1.0545

80.4544

014

himborazo

umanda

2.2050

79.1356

014

l Oro

achala

3.2586

79.9605

014

smeraldas

orbón

.0883

78.9889

020

estern Andean

lopes

anto

omingo de

os Tsáchilas

anto

omingo

0.2530

79.1753

014

mazon basin

apo

ena

0.9938

77.8128

014

ucumbios

ago Agrio

.0860

76.8952

014

rellana

rancisco de

rellana

0.4664

76.9871

014

astaza

uyo

1.4836

78.0025

014

alápagos

anta Cruz

7.7862

63.1811

014

Aedes albopictus

Region

Province

Locality

La�tude

Longitude

Collec�on

ear

Number of

pecimens

aciﬁc coast

mbabura

achaco

.8333

78.4666

020

Table 1. Collecon sites of Aedes aegyp and Aedes albopictus individuals

in tropical and subtropical regions of Ecuador.

Código ISSN 2588-0551

https://www.inspilip.gob.ec

Parasitism of Ascogregarina culicis (Apicomplexa: Lecudinidae) in immature stages and adults of Aedes aegypti

Carrazco Montalvo A

Magazine INSPILIP - V 6 - Number 1 - May 2022

Código ISSN 2588-0551

Parasite morphological identication





tubules were removed and placed on a slide and

stained for 30 seconds with Lugol’s solution. The

  Ascogregarina at each stage was

performed and photographed using an optical

      



DNA extraction, PCR, and sequencing



macerated using a micropistile. DNA was extracted

using the DNeasy Blood & Tissue Kit (Qiagen,



     

overnight at 56°C and genomic DNA was eluted in





ribosomal RNA (ssrRNA) was performed using the







The PCR amplicons obtained by RPLU primers were

      

services (Seoul, South Korea).

      

BLAST database (Basic Local Alignment Search

      

Ascogregarina genus. For genetic analysis, sequence

alignment and cleaning were performed using





in the NCBI repository.

Results

The microscopy and molecular analysis showed the

infection by Ascogregarina in 30 individuals Ae.

aegypti       

stages of Ascogregarina culicis   



located in the extracellular tissue of the intestine

(Figure 1a).

While the pupae had gametocytes of the parasite

      

the adults presented oocysts located also in the



The parasite was not found in Ae. albopictus

      

        

Ascogregarina.

The BLAST result indicated 100% identity and

query cover for Ascogregarina culicis 



Ascogregarina culicis was detected in Ae. aegypti from

     

  



Aedes aegypti infected with Ascogregarina culicis

were collected during the dry, rainy, and transition

      

       

mosquitoes from Guayaquil. We present a summary

of the individuals collected and the infection rate



Código ISSN 2588-0551

Parasitism of Ascogregarina culicis (Apicomplexa: Lecudinidae) in immature stages and adults of Aedes aegypti

Carrazco Montalvo A

Código ISSN 2588-0551

https://www.inspilip.gob.ec

Magazine INSPILIP - V 6 - Number 1 - May 2022

Figure 1. Ascogregarina culicis found in diﬀerent stages of Aedes aegyp.

a. Trophozoite in the fourth larvae stage in the mid-gut of the larvae.

b. Gametocytes in the Malpighian tubules of pupae.

c. Oocyst in the Malpighian tubules of adults.

THE MOSQUITO AND PARASITE

LIFE CYCLE

Adult

pupse

4 th

inster larvae

2 nd and 3 rd

inster larvae

1 st

inster larvae

Figure 2. Map of distribuon of Ascogregarina culicis in Aedes aegyp in Ecuador.

This shows posive and negave samples.

Ae. aegyp nega�ve for

Ascogregarine culis

Aedes albopictus nega�ve for

Ascogregarine culis

16 Cachaco

1 Santa Cruz

2 Esmeraldas

3 Borbon

4 Manta

5 Guayaquil

6 Babahoyo

7 Chone

8 portoviejo

9 Cumandá

10 Santo Domingo

11 Machala

12 Tena

13 Lago Agrio

14 Orellana

15 Pastaza

100 0 100 200km

Aedes aegyp posi�ve

for Ascogregarine culis

Código ISSN 2588-0551

https://www.inspilip.gob.ec

Parasitism of Ascogregarina culicis (Apicomplexa: Lecudinidae) in immature stages and adults of Aedes aegypti

Carrazco Montalvo A

Magazine INSPILIP - V 6 - Number 1 - May 2022

Código ISSN 2588-0551

Discussion

      Ascogregarina culicis

infecting Aedes aegypti in Ecuador. Previous studies

have reported Ae. aegypti infected with this parasite





The pathogenicity of Ascogregarina on the larval

development of Ae. aegypti has contrasting reports.

Some authors have reported higher mortality and

shorter larval development



, while others have

        



Nevertheless, the possibility of using Ascogregarina

as a biocontrol agent has been suggested, mainly

      

conditions (high dosage infection, crowding,

     

nutrients), the mortality rate of mosquito larvae is

increased



Ascogregarina culicis and Ascogregarina taiwanensis



have been proposed as biological control of Ae.

aegypti and Ae. albopictus, respectively



In this study, Ascogregarina culicis was recorded

during the rainy, dry, and transition seasons in a wide

range of areas in Ecuadorian Coastal and insular

regions. This has been previously reported in other

studies

, where the abundance of Ascogregarina

parasite is correlated with high numbers of Aedes

mosquitoes as its main reservoirs. Additionally,

the molecular analysis of the parasite showed

polymorphism in a sample from Esmeraldas that

indicates genetic variability



Ascogregarina parasites were not detected in samples





   Ascogregarina using

     

because Ascogregarina culicis and Ascogregarina





process, reducing the subjectivity of morphological

techniques



For this analysis, a protocol initially designed to

identify Plasmodium was used

, since Plasmodium

and Ascogregarina belong to the same phylum.

        



Table 2. Aedes aegyp with the presence of Ascogregarina culicis

idenﬁed by microscopy and PCR

Esmeraldas

01/09/2014 Dry

3/23 (13 %)

16/06/2014 Transi�on

Guayaquil

17/03/2014

12/68 (18 %)

31/03/2014 Rainy

19/05/2014 Rainy

09/06/2014 Transi�on

Manta

23/06/2014 Transi�on

1/8 (13 %)

Santa Cruz

24/08/2014 Dry

1/9 (11 %)

Borbón 10/03/2020 Rainy 13 13/95 (14 %)

Total

30/260 (12 %)

Locality Collec�on date - Season

Parasite posi�ve

mosquitoes

Infec�on rate

Código ISSN 2588-0551

Parasitism of Ascogregarina culicis (Apicomplexa: Lecudinidae) in immature stages and adults of Aedes aegypti

Carrazco Montalvo A

Código ISSN 2588-0551

https://www.inspilip.gob.ec

Magazine INSPILIP - V 6 - Number 1 - May 2022





not as reliable and molecular data are fragmentary



     Ascogregarina

parasites on Aedes



naturally infecting parasite.

There may be a relationship between the presence

of Ascogregarina and arboviral infections, mainly

because the mosquito midgut epithelium cells are

used by Ascogregarina   

to complete the parasite life cycle or complete

replication, respectively.

      

Ascogregarina on pathogen transmission by





suggested

      

transmitted via Ascogregarina oocysts in Ae. aegypti.

On the other hand, a recent study by Thongsripong

and Wesson



concluded that Ascogregarina culicis



virus infection in Ae. aegypti.

Aedes albopictus      

the Cachaco locality and all the specimens were

negative for Ascogregarina species. Several articles

report Ae. albopictus infected by Ascogregarina

taiwanensis



, and it has been suggested that

Ae. albopictus has a competitive advantage in the

absence of parasitism



Although there are contrasting criteria among authors

regarding the potential of Ascogregarina as mosquito

biocontrol and as an arboviral prevention agent, our

     

      

replication and transmission.

Conicts of Interest: 

of interest.

Acknowledgments:    

Francesca Ponce, and Stephany Villota, for revising

the manuscript.

Funding:

Institutes of Health – National Institute of Allergy and



      



    

     

    



Data Availability Statement: Sequences used in this





References

         



        

genomics reveals that an anthropophilic population

Aedes aegypti mosquitoes in West Africa recently

gave rise to American and Asian populations of this

       



     

of mosquitoes: In Integrated mosquito control





        

Fly Gregarines of the Genus

Ascogregarina and

Psychodiella (Apicomplexa: Eugregarinorida,

Aseptatorina) – Overview of Their Taxonomy, Life

     

      



        





Ascogregarina species

(Apicomplexa: Lecudinidae) in

Aedes aegypti and

Aedes albopictus (Diptera: Culicidae) by polymerase

       



5. Thongsripong, P., Chandler, J. A., Green, A. B.,

Kittayapong, P., Wilcox, B. A., Kapan, D. D.,

     

    

symbionts across habitat types in Thailand endemic

     

   

ece3.3676



First Report of

Aedes (Stegomyia) albopictus

     

      



tjx165

Código ISSN 2588-0551

https://www.inspilip.gob.ec

Parasitism of Ascogregarina culicis (Apicomplexa: Lecudinidae) in immature stages and adults of Aedes aegypti

Carrazco Montalvo A

Magazine INSPILIP - V 6 - Number 1 - May 2022

Código ISSN 2588-0551

         

Ascogregarina taiwanensis and Ascogregarina

culicis (Apicomplexa: Lecudinidae) in larvae of

Aedes albopictus and Aedes aegypti (Diptera:

     

      



          





Ascogregarina taiwanensis infection

Aedes aegypti and Aedes albopictus in Santa



  



         

          

    

Aedes albopictus and Aedes aegypti from



  



        

Pinheiro, L., do Rosario V. E., Thaithong, S., Brown,



       

      

  





geneious.com

        

Ascogregarina culicis in Temperate Argentina:

   

Aedes aegypti Larvae

and Pupae. J. Invertebr. Pathol. 101 (3). Elsevier Inc.:



       

Seasonality of

Ascogregarina culicis (Apicomplexa:

Lecudinidae) in Natural Populations of

Aedes aegypti

(Diptera: Culicidae) from Temperate Argentina.

      



    

     

Aedes

      





Susceptibility of

Aedes aegypti and Aedes albopictus

larvae to

Ascogregarina culicis and Ascogregarina

taiwanensis (Apicomplexa: Lecudinidae). Florida. J





          

   

Culicis and Aedes

Aegypti      





Controlled by Infectious Dose Combinations and







     





      



      

Ascogregarina taiwanensis. Edited by Olle Terenius.





         

Ascocystis (Apicomplexa, Lecudinidae) from





      

     

Aedes

aegypti mosquitoes across Thailand based on nuclear





Parasitism of Ascogregarina culicis (Apicomplexa: Lecudinidae) in immature stages and adults of Aedes aegypti

Carrazco Montalvo A

Código ISSN 2588-0551

https://www.inspilip.gob.ec

Magazine INSPILIP - V 6 - Number 1 - May 2022



K.V.; Schrevel, J.; Aleoshin, V.V. A new view on the

morphology and phylogeny of eugregarines suggested

by the evidence from the gregarine Ancora sagittata

    

     









Ascogregarina culicis (Apicomplexa: Lecudinidae)

    

      



       

Ascogregarina culicis (Eugregarinorida:

      

Infection in

Aedes aegypti (Diptera: Culicidae),





       

Ascogregarina



    





of infection, development and gametocyst formation

Ascogregarina taiwanensis (Apicomplexa:

Lecudinidae) in its mosquito host,

Aedes albopictus

      

 





Aedes albopictus by Ascogregarina taiwanensis

lowers its competitive ability against

Aedes triseriatus.

    

