Susceptibility of Aedes aegypti (Diptera: Culicidae) to Acanthamoeba polyphaga (Sarcomastigophora: Acanthamoebidae)

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Parasitol Res (2010) 107:195–198 DOI 10.1007/s00436-010-1843-9


Susceptibility of Aedes aegypti (Diptera: Culicidae) to Acanthamoeba polyphaga (Sarcomastigophora: Acanthamoebidae) Marilise Rott & Karin Caumo & Ismael Sauter & Janina Eckert & Luana da Rosa & Onilda da Silva

Received: 23 December 2009 / Accepted: 11 March 2010 / Published online: 9 April 2010 # Springer-Verlag 2010

Abstract To date there is no report on mosquitoes infected with free-living amoebae. For this reason, the aim of this study was to verify if Aedes aegypti could be susceptible to Acanthamoeba polyphaga under laboratory conditions, so trophozoites were offered as a unique food resource for larvae of first instar. The results show that those amoebae are able to infect and colonize the mosquito gut and could be re-isolated of all stages of the mosquito (larvae, pupae, and adults).

Introduction Free-living amoebae (FLA) belong to the genus Acanthamoeba, are found worldwide, and inhabit a great variety of natural environments. These amoebae also are able to live endozoically in diverse hosts, and for this reason, they are designated as amphizoic microorganisms (Visvesvara et al. 2007). They have been isolated from the soil, salt and freshwater, humans, and several domestic and wild animals (Marciano-Cabral et al. 2000; Pens et al. 2008; Caumo et al. 2009; Carlesso et al. 2009). The genus Acanthamoeba is the most common among the FLA (Page 1988); however, only some species are pathogenic to humans, with the potential to cause granulomatous amoebic encephalitis, cutaneous lesions, and sinusitis in immunocompromised M. Rott (*) : K. Caumo : I. Sauter : J. Eckert : L. da Rosa : O. da Silva Microbiologia Imunologia e Parasitologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil e-mail: [email protected]

patients and amoebic keratitis in immunocompetents associated especially with contact lense wearers (Khan 2006). Acanthamoeba have adapted to resist these diverse conditions by switching their phenotype. Under harsh environmental conditions such as lack of nutrients, high temperatures, and high osmolarity, this protozoan change into a resistant cyst form. Under favorable conditions, those cysts change into vegetative and infective trophozoite forms, completing life cycle. It is known that some species of protozoan infect mosquitoes, causing a diversity of diseases. For instance, some species of Ascogregarina are known to parasite Aedes aegypti and Aedes albopictus (Huang et al. 2006; Roychoudhury and Kobayashi 2006; Roychoudhury et al. 2007; Dos Passos and Tadei 2008; Albicócco and Vezzani 2009). A. aegypti is found throughout the world in tropical and subtropical regions, mainly in urban and suburban areas. This mosquito is the principal vector of dengue and dengue hemorrhagic fever. Since there is no vaccine for these arboviral diseases, mosquito control is the only method available (WHO 1997). Currently, three different methods can be used to control A. aegypti: the elimination of larval habitats (flower pots, rain gutters, abandoned tires, water storage tanks), chemical control, and biological control. Studies (Polson et al. 2002; Rodriguez et al. 2003) have shown that repeated pesticide applications produce a negative impact on the environment as well as the development of resistance in larvae and adults. Biological control could be an alternative approach to avoid these effects. Some predators such as fish (Martinez-Ibara et al., 2002), copepods (Cyclopoidae) (Marten et al. 1994; Schreiber et al. 1996), and notonectids (Chesson 1984)


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Fig. 1 Negative control, midgut of fourth instar larvae of A. aegypti (left). Midgut of fourth instar larvae of A. aegypti infected with A. polyphaga (right)

have been evaluated as potential control agents of A. aegypti larvae. Bacillus thuringiensis (Chen et al. 2009) and Romanomermis culicivorax (Martinez-Ibara et al. 2002, 1998; Santamarina et al. 2000) have been assessed as a potential biocide of A. aegypti. So far, there are no records of A. aegypti larvae parasitized by Acanthamoeba. In view of the plausibility of Acanthamoeba spp. to be used in biological control of mosquitoes, we investigated if A. aegypti could be susceptible to infection with Acanthamoeba polyphaga.

Material and methods Amoeba cultivation The study was performed using trophozoites of A. polyphaga (ATCC30872) isolated from freshwater. The isolates were cultured axenically in PYG medium [proteose peptone 0.75% (w/v), yeast extract 0.75% (w/v), and glucose 1.5% (w/v)] at 30°C. For the bioassays, axenic cultures containing 106 to 107 trophozoites/mL were centrifuged for 5 min at 350×g. The supernatant was discarded, and the precipitate was washed twice with phosphate-buffered saline (PBS). The precipitate of amoebae was diluted in Page's saline to obtain a concentration of 1×106 trophozoites/mL.

Larval gut dissection A pool of twenty healthy larvae from different instars (second to fourth) as well as pupae and adults were examined for the presence of A. polyphaga trophozoites and cysts. The bioassays were carried out through the gut dissection in 0.9% NaCl physiological solution, according to the techniques of Beier and Craig (1985) and Garcia et al. (1994). The dissected guts were examined for the presence of cysts and trophozoites under an optical microscope using ×10, ×40, and ×100 magnification. Those cysts and trophozoites were identified based on morphological aspects, according to Schuster and Visvesvara (2004). After that, the guts were macerated and submitted to A. polyphaga isolation in non-nutrient agar (NNA) plates containing an overlayer of Escherichia coli. Re-isolation of A. polyphaga A pool of larvae, pupae, and adults (20 per bioassay) were washed three times in PBS and macerated in 1 mL distilled sterile water. The precipitate obtained was transferred to 1.5 % NNA plates containing an overlayer of heat-inactivated (56°C/2 h) E. coli (ATCC 25922) suspension. The plates were sealed with Parafilm® and incubated at 30°C for up to 15 days. Three plates were prepared for each sample and

Infection of A. aegypti Eggs of a laboratory-reared A. aegypti (Rockfeller strain) were placed on container containing distilled sterile water (300 mL). After hatching, larvae of first instar were distributed in five glass containers (at least 20 larvae per container), and 100 μL of the trophozoites suspension of A. polyphaga were offered as a unique food resource for a period of 24 h. After this time, the larvae fed only on puppy food until the development is complete. As a negative control, larvae free of trophozoites received only puppy food. Larval susceptibility of A. aegypti to A. polyphaga was determined under 25°C and photophase, 12 h in an incubator (Tecnal®).

Fig. 2 Trophozoite of A. polyphaga found in the midgut of fourth instar larvae. Magnifications of ×100

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were examined daily by means of an optical microscope (×100) to check the presence of A. polyphaga.

Results The study of experimental infection of A. polyphaga in A. aegypti indicated that these amoebae are able to infect and develop in this species of mosquito. Cysts and trophozoites of A. polyphaga were observed in all larval instar, in pupae, and adults. In the control group, forms of A. polyphaga were found (Figs. 1 and 2). The infection was confirmed after re-isolation of A. polyphaga from the macerated intestines dissected on plates of agar with Non-Nutrient Agar (NNA) E. coli in triplicate. Positive results for infection of A. polyphaga were also found when pools of larvae, pupae, and adults were macerated and further processed into plates ANN with E. coli.

Discussion As exposed, A. polyphaga are able to develop and reproduce in the gut of A. aegypti. When larvae, pupae, and adults were dissected, endosymbiont bacteria were observed. It is known that mosquitoes carry a variety of microbial endosymbionts (Azambuja et al. 2005; Rani et al. 2009). Such organisms could serve as nutrition for A. polyphaga to survive in the gut of A. aegypti. Since this Acanthamoeba strain was re-isolated from larval stages, pupae, and adults of A. aegypti, we believe that this organism is able to escape the mechanism proposed by Moncayo et al. (2005). These authors described that peritrophic membrane in larval mosquitoes is a very robust structure and serves to prevent or reduce pathogen invasion. By enveloping the meconium, the meconial peritrophic matrices would sequester microorganisms ingested during the larval stage along with the meconium. How this mechanism occurs in A. aegypti needs to be studied. To date, it is unknown if A. aegypti do naturally carry Acanthamoeba. For this reason, we are collecting field specimens to determine infection rates. We are also studying which part of the mosquito the protozoan develops. Parameters that could explain any effect of these parasites on such hosts were not evaluated. The mosquito– Acanthamoeba interaction could provide new information about the use of these organisms as biological agents for vector-control strategies, since it is known that these amoeba serves as a reservatory of different microorganisms. Furthermore, the use of Acanthamoeba pathogenic strains could kill or even produce life cycle alteration in A. aegypti,


as observed with other organisms like gregarines (Fukuda et al. 1997; Reyes-Villanueva et al. 2003). Our studies report, for the first time, infection of A. aegypti by Acanthamoeba, then details of these host– parasite relationship have to be investigated.

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