Description of the ovarian microbiota of Aedes aegypti (L) Rockefeller strain

Highlights

• Ovarian microbiota is modified by the physiological changes during blood feeding

• Ovarian microbiota during blood feeding becomes specifically hemolytic

• Serratia marcescens potentially intervenes in the process of blood digestion

• Some bacteria identified (Serratia, Pantoea, Pseudomonas) have a biotechnological potential against parasites and viruses.

Abstract

Aedes aegypti is one of the vectors responsible for transmitting the viruses that cause dengue, Zika and chikungunya in the human population. Mosquitoes have bacterial communities in different organs, mainly in the midgut, but to a lesser extent in their reproductive organs, such as the ovaries, where replication and vertical transmission is decisive for dengue virus. These bacteria also influence metabolic and physiological processes such as ingestion and digestion of blood. In this study, aerobic bacterial communities associated with ovaries of A. aegypti Rockefeller strain were determined, describing their potential function during ovocitary development. The groups of mosquitoes were separated into three treatments: diet with 10% sugar solution, diet with blood supply, and blood feeding combined with tetracycline. The ovaries were extracted from the mosquitoes, and then put in enriched culture media (blood and nutritive agar) by direct inoculation, for subsequent isolation and macroscopic and microscopic characterization of the colonies. The taxonomic determination of bacterial isolates was achieved by sequence analysis of the 16S rRNA gene. A higher bacterial load was observed in the sugar feeding group (6 × 10³ CFU/ml) in contrast to the group fed only with blood, with and without an antibiotic (4.03-4.04 × 10³CFU/ml; 4.85-5.04 × 10³CFU/ml). As a result, a total of 35 colonies were isolated, of which 80% were gram-negative and 20% gram-positive; 72% were lactose negative and 8% lactose positive. Of the total bacteria, 83% had gamma hemolysis, 17% alpha hemolysis, and none presented beta hemolysis. After phenotypic and biochemical characterization, 17 isolates were selected for molecular identification. Only phyla Actinobacteria and Proteobacteria were found. Bacteria associated with ovaries of A. aegypti were mainly identified as belonging to the Serratia and Klebsiella genera. Some bacteria (Serratia marcescens, Pantoea dispersa and Klebsiella oxytoca) have wide biotechnological potential due to their entomopathogenic power and their bioactivity against different pathogens.

Introduction

Aedes aegypti is a highly anthropophilic mosquito that is present in tropical and subtropical regions and is the main vector of arboviral diseases such as Dengue, Zika, and Chikungunya, which presents a huge risk to public health (World Health Organization 2009; Raharimalala et al., 2012). These mosquitoes can harbor a great diversity of microorganism communities including mostly Proteobacteria, Bacteroidetes, and Actinobacteria phyla (Minard et al., 2013; Mancini et al., 2018). These phyla can fulfill important functions in metabolic and physiological processes for the insect, like nutrition, development, behavior, homeostasis, carbohydrate and protein synthesis, reproduction, and immune system modulation (Hillyer 2004; Gaio et al., 2011; Charan et al., 2016; Kumar et al., 2018).

Studies in A. aegypti have revealed the richness of the microbiota associated with different organs such as the midgut, and point to the bacteria of Serratia sp. and Enterobacter sp. genera, for affecting some physiological processes, as the change of pH in the host during the process of blood ingestion and blocking the arbovirus (Gusmão et al., 2010; Zouache et al., 2011; Yadav et al., 2015). In this context, many bacterial species (e.g., Wolbachia, Asaia) may also have the possibility of modulating the vector competence of A. aegypti to reduce the ability to transmit pathogens to humans, for which the Wolbachia endosymbiont has been used in biological control strategies at the continental level (Hoerauf and Rao 2007; Ye et al., 2013). Wolbachia is able to invade and maintain themselves in the arthropod species through manipulation of the host's reproduction.

Despite the generation of knowledge about the microbiota in A. aegypti focused on key organs such as the midgut, there are few studies on the microbiota associated with other important organs, such as the salivary glands and the reproductive organs (ovaries). Specifically, the ovaries are of interest because they are virus replication sites and because the presence of certain bacteria may influence the establishment and displacement of other symbionts with demonstrated biotechnological and biological power for the vector control of A. aegypti (Ricci et al., 2012; Minard et al., 2013; Mancini et al., 2018). The few existing studies there are indicate that the bacterial load and richness of the salivary gland and ovaries is lower compared to the midgut (Gaio et al., 2011). Furthermore, the midgut has been documented as having a higher frequency of Asaia and Pantoea genera as strictly associated bacteria in these organs of A. aegypti (Gaio et al., 2011).

Therefore, it is necessary to describe in greater detail the composition and dynamics of the microbiota of the ovaries and its potential function in the body. Specifically, establishing microbial biomarkers of the productive structures associated with different states of feeding, infection or age of A. mosquitoes is also of great relevance, because they can be determinant in fitness and copulation preferences (Brun et al., 1974; Ponnusamy et al., 2008). Ovaries correspond to a space where a determinant source of proteins for the development of oocytes is supplied, and that during their development, structural, and physiological changes for the life cycle and survival of Aedes are generated (Gaio et al., 2011; Gonzales et al., 2015).

The biological potential of the microbiota associated with the ovaries of A. aegypti is understudied. Therefore, describing the mosquito-microbiota interaction could support new methods of biological control of the vector. These microbiotas can even be used for the development of paratransgenesis strategies due to their paternal and maternal transmission capacity in A. aegypti. This research is a baseline study that provides new knowledge on the microbial (specifically bacterial) communities associated with aerobic culturable fraction of ovaries in A. aegypti Rockefeller strain under different feeding regimes using culture-dependent methods. In addition, the study by classical methods of microbiology produces an inventory of bacteria that can be used in bioprospecting, as well as trials of antiviral activity against different arboviruses because the ovaries are a space of constant interaction with them. Bacterial isolation thus becomes a source for secondary metabolite searching.