Replacing a native Wolbachia with a novel strain results in an increase in endosymbiont load and resistance to dengue virus in a mosquito vector

Abstract

Wolbachia is a maternally transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce pathogen interference and spread into mosquito vector populations makes it possible to develop Wolbachia as a biological control agent for vector-borne disease control. Although Wolbachia induces resistance to dengue virus (DENV), filarial worms, and Plasmodium in mosquitoes, species like Aedes polynesiensis and Aedes albopictus, which carry native Wolbachia infections, are able to transmit dengue and filariasis. In a previous study, the native wPolA in Ae. polynesiensis was replaced with wAlbB from Ae. albopictus, and resulted in the generation of the transinfected "MTB" strain with low susceptibility for filarial worms. In this study, we compare the dynamics of DENV serotype 2 (DENV-2) within the wild type "APM" strain and the MTB strain of Ae. polynesiensis by measuring viral infection in the mosquito whole body, midgut, head, and saliva at different time points post infection. The results show that wAlbB can induce a strong resistance to DENV-2 in the MTB mosquito. Evidence also supports that this resistance is related to a dramatic increase in Wolbachia density in the MTB's somatic tissues, including the midgut and salivary gland. Our results suggests that replacement of a native Wolbachia with a novel infection could serve as a strategy for developing a Wolbachia-based approach to target naturally infected insects for vector-borne disease control.

Figure 1. Inhibition of dengue infection in the whole bodies of MTB mosquitoes.

At 14 dpi (days post infection) through a blood meal or intrathoracic injection, the whole bodies of MTB and APM mosquitoes were collected, and the number of genome copies of the DENV genome was determined by qRT-PCR using primers for the NS5 gene; the results were normalized to the Ae. polynesiensis ribosomal protein S6 (RPS6). Lines indicate the median of the ten biological replicates. Significance was determined using a Mann-Whitney U test.

Figure 2. Inhibition of dengue infection in the midgut of MTB mosquitoes.

(A) At 4, 7 and 10 dpi with a blood meal containing DENV-2, mosquito midguts were collected, and the number of genome copies of the DENV-2 genome was determined by qRT-PCR using primers for the NS5 gene; the results were normalized to the Ae. polynesiensis RPS6. Lines indicate the median of the ten biological replicates. Significance was determined using a Mann-Whitney U test. (B) At 7 dpi through a blood meal, mosquito midguts were collected and fixed, and the viral antigen was detected using a mouse anti-Dengue complex monoclonal antibody by indirect fluorescence assay (IFA). One representative midgut from each category is shown. APM mosquitoes fed with dengue-infected or -uninfected blood meal was used as positive (POS.) and negative (NEG.) control, respectively. +, dengue-positive; −, dengue-negative.

Figure 3. In vitro assay of DENV-2 transmission by MTB and APM mosquitoes.

At 14 days after a blood meal containing DENV-2 or infection through intrathoracic injection, the wings and legs were removed, and the proboscis of each mosquito was inserted into a feeding solution for 90 min. Solution from each mosquito was analyzed for infectious DENV-2 by plaque assays. Lines indicate the log of the median values. Twenty eight and eight biological replicates were used in oral feedings and intrathoracic injection assays, respectively.

Figure 4. Resistance of MTB mosquitoes to DENV-2 is associated with a high density of Wolbachia in mosquito somatic tissues.

The fold change in genome copy of the Wolbachia surface protein (WSP) gene in MTB mosquitoes is compared to APM mosquitoes. The copy number of the Wolbachia wsp was normalized by Ae. polynesiensis RPS6. In all the assays, the midguts, salivary glands, fat bodies, and ovaries of 7-day-old non-blood-fed females were dissected and used for extraction of total genomic DNA. Error bars are standard errors of the mean of twelve biological replicates.