Vectorial Capacity of Aedes aegypti for Dengue Virus Type 2 Is Reduced with Co-infection of Metarhizium anisopliae

et al. (2013) Vectorial Capacity of Aedes aegypti for Dengue Virus Type 2 Is Reduced with Co-infection of Metarhizium anisopliae. PLoS Negl Trop Dis 7(3): e2013. doi:10.1371/journal.pntd.0002013 Vectorial Capacity of Aedes aegypti for Dengue Virus Type 2 Is Reduced with Co-infection of Metarhizium anisopliae Javier A. Garza-Herna ndez 0 Mario A. Rodrguez-Pe rez 0 Ma Isabel Salazar 0 Tanya L. Russell 0 Monsuru A. Adeleke 0 Erik de J. de Luna-Santillana 0 Filiberto Reyes-Villanueva 0 Michael J. Turell, U.S. Army Medical Research Institute of Infectious Diseases, United States of America 0 1 Laboratorio de Biomedicina Molecular, Centro de Biotecnolog a Geno mica, Instituto Polite cnico Nacional , Reynosa, Tamaulipas , Mexico , 2 Laboratorio de Inmunolog a Celular e Inmunopatoge nesis, Departamento de Inmunolog a, Escuela Nacional de Ciencias Biol o gicas, Instituto Polite cnico Nacional , Mexico City, Distrito Federal , Mexico , 3 Faculty of Medicine, Health and Molecular Sciences, James Cook University , Cairns, Queensland , Australia , 4 Public Health Entomology and Parasitology, Department of Biological Sciences, Osun State University , Osogbo , Nigeria Background: Aedes aegypti, is the major dengue vector and a worldwide public health threat combated basically by chemical insecticides. In this study, the vectorial competence of Ae. aegypti co-infected with a mildly virulent Metarhizium anisopliae and fed with blood infected with the DENV-2 virus, was examined. Methodology/Principal Findings: The study encompassed three bioassays (B). In B1 the median lethal time (LT50) of Ae. aegypti exposed to M. anisopliae was determined in four treatments: co-infected (CI), single-fungus infection (SF), singlevirus infection (SV) and control (C). In B2, the mortality and viral infection rate in midgut and in head were registered in fifty females of CI and in SV. In B3, the same treatments as in B1 but with females separated individually were tested to evaluate the effect on fecundity and gonotrophic cycle length. Survival in CI and SF females was 70% shorter than the one of those in SV and control. Overall viral infection rate in CI and SV were 76 and 84% but the mortality at day six post-infection was 78% (54% infected) and 6% respectively. Survivors with virus in head at day seven post-infection were 12 and 64% in both CI and SV mosquitoes. Fecundity and gonotrophic cycle length were reduced in 52 and 40% in CI compared to the ones in control. Conclusion/Significance: Fungus-induced mortality for the CI group was 78%. Of the survivors, 12% (6/50) could potentially transmit DENV-2, as opposed to 64% (32/50) of the SV group, meaning a 5-fold reduction in the number of infective mosquitoes. This is the first report on a fungus that reduces the vectorial capacity of Ae. aegypti infected with the DENV-2 virus. - . These authors contributed equally to this work. The susceptibility of Aedes aegypti adults to infection with Beauveria bassiana was first reported in the late 1960s [1]. However the potential of entomopathogenic Ascomycetes (Hypocreales) as adulticides of vector mosquitoes was largely overlooked until Metarhizium anisopliae was demonstrated to induce mortality of Culex quinquefasciatus and Anopheles gambiae [2]; and sequentially both M. anisopliae and B. bassiana have been tested against Ae. aegypti and Aedes albopictus [3]. The successful infection of adult female mosquitoes has been made via direct contact [4,5] and also via auto-dissemination from males to females when mating [6,7]. The increasing interest in exploring these fungi as biocontrol agents of dengue vectors stems from the fact that they are ubiquitously available, relatively cheap to mass-produce, and kill mosquitoes effectively [8]. In addition to the infection studies, attention has also been focused on other topics such as determining their safety to public health [9], and the effect of different surfaces on the infectivity of conidia to resting mosquitoes [10]. Likewise, some devices with inoculum baited with lures have also been tested for attracting and infecting adults to avoiding domiciliary sprayings [11]. Metarhizium anisopliae pathogenesis to insects has been widely documented [12]. The fungus is hemibiotrophic [13]. Conidia germination and cuticle perforation last around 24 hours [14]. After penetration, the pathogen produces hyphal bodies or blastospores invading the whole hosts hemocele, depleting nutrients and killing the insect by starvation, dehydration, and toxemia [15]. It is therefore proposed that the rapid fungal invasion could affect the survival of the DENV virus if both are present in the same female of Ae. aegypti, weakening its vectorial competence. Here, we fed Ae. aegypti females with DENV-2-infected human blood, and/or Dengue is a worldwide public health problem. There is not an effective vaccine yet; the chemical struggle against its transmitter, the mosquito Aedes aegypti, is onerous and erratic, and the community participation to eliminate vector breeding sites is unconfident. Here, we examined mosquitoes fed on human blood mixed with the Dengue virus, by exposure to the fungus Metarhizium anisopliae, to test whether the fungus halts the viral dissemination from midgut to head in co-infected (CI) insects. We found an overall viral infection rate in CI mosquitoes of 76% but infected or not, most (78%) died before or at day six postinfection; only six (12%) out of 50, survivors had virus in head and were potentially infectious at day seven postinfection. A higher infection (84%) was observed in singlevirus infected mosquitoes, but they suffered only 6% mortality after 6 days and 32 (64%) survivors tested positive for virus in head after 7 days. Survival, fecundity and ovaric cycle of CI mosquitoes were reduced in 70, 52 and 40% in comparison to the ones of control. Therefore, if the fungus caused a 5-fold reduction in the number of infectious mosquitoes, it has potential to be evaluated against the Dengue transmitter in field. exposed them to M. anisopliae conidia to produce single-fungus (SF), single-virus (SV) and co-infected (CI) mosquitoes. The parameters evaluated included mosquito survival, fecundity and first gonotrophic cycle (GC) length, plus the viral infection rate in the midgut and head. Materials and Methods Cell lines, virus titers, fungus and mosquito infection The DENV-2 Yuc 18500 strain was isolated from blood of a sick person at Merida city in 2008; it is deposited at the Collection of Arboviruses isolated at the Yucatan Peninsula of the Regional Research Center Dr Hideyo Noguchi, University of Yucatan (UADY), Mexico; its use in this study was approved by written consent given by Dr. Fernando Andrade-Narvaez, Chair, Bioethics Committee of the Regional Research Center Dr Hideyo Noguchi, University of Yucatan (UADY), Merida, Yucatan, Mexico. In addition, all members of the Bioethics Committee provided informed consent. This strain was used to infect C6/ (...truncated)