Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector

Abstract

Here, we show that an alpha-proteobacterium of the genus Asaia is stably associated with larvae and adults of Anopheles stephensi, an important mosquito vector of Plasmodium vivax, a main malaria agent in Asia. Asaia bacteria dominate mosquito-associated microbiota, as shown by 16S rRNA gene abundance, quantitative PCR, transmission electron microscopy and in situ-hybridization of 16S rRNA genes. In adult mosquitoes, Asaia sp. is present in high population density in the female gut and in the male reproductive tract. Asaia sp. from An. stephensi has been cultured in cell-free media and then transformed with foreign DNA. A green fluorescent protein-tagged Asaia sp. strain effectively lodged in the female gut and salivary glands, sites that are crucial for Plasmodium sp. development and transmission. The larval gut and the male reproductive system were also colonized by the transformed Asaia sp. strain. As an efficient inducible colonizer of mosquitoes that transmit Plasmodium sp., Asaia sp. may be a candidate for malaria control.

Fig. 1.

Images of Asaia sp. from An. stephensi. (A) Colonies of Asaia sp. on CaCO₃-agarized medium, showing a characteristic pink color. (B) Haloes of carbonate solubilization due to acidification. (C) TEM micrograph of an An. stephensi adult midgut full of bacteria that probably belongs to genus Asaia. (D and E) Magnification of bacterial cells in the midgut (D), whose morphology resembles that of Asaia sp. in pure culture (E). Filamentous structures in the nucleoid region (asterisks), and an extracellular matrix with a fibrillar nature (arrow) can be noted. Cells of Asaia sp. in pure culture do not show the extracellular matrix observed in the mosquito midgut. (F) The midgut lumen of An. stephensi is full of bacterial cells (arrow). (G) ISH with an Asaia-specific probe showing a high density of the bacterium in the midgut lumen (arrows).

Fig. 2.

Recolonization of the adult An. stephensi gut by a Gfp-tagged Asaia. Phase contrast (A and C) and fluorescence (B and D) microscope images of Asaia sp. strain SF2.1(Gfp), recolonizing An. stephensi midgut. Male (A and B) and female (C and D) terminal portions of the midgut. Malpighian tubules are visible (red arrows). (E and F) Midgut terminal portion of a female fed with sucrose solution without Asaia sp. strain SF2.1(Gfp). (G and H) Laser scanning confocal microscope images of adult mosquito guts, showing high concentrations of strain SF2.1(Gfp) microcolonies.

Fig. 3.

Colonization of male gonads of adult An. stephensi by Asaia sp. A large number of Asaia cells was found in the reproductive organs of adult, male An. stephensi. (A–D) Reproductive organs of an An. stephensi adult male fed (A and B) or not fed (C and D) with a sucrose-containing solution in which Asaia sp. SF2.1(Gfp) cells were suspended. A testis (asterisk) and initial portion of a gonoduct (arrow) are visible by phase contrast (A and C) and fluorescence (B and D) microscopy. Fluorescent Asaia sp. SF2.1(Gfp) cells are visible in the testis and in the gonoducts. (E–G) Male gonoduct, after colonization by Asaia sp. strain SF2.1(Gfp), visualized by fluorescent (E), phase contrast (F), and laser scanning confocal microscopy (G). The images indicate a high concentration of Asaia sp. is in the gonoduct. (H) Higher magnification of a microcolony of Asaia sp. strain SF2.1(Gfp) in the male gonoduct. (I) ISH showing cell clusters of Asaia sp. cells (circles) colonizing the male reproductive organs. Asterisks indicate spermatic bundles. A sperm duct (open black arrow) is visible. Intestine is indicated by a black arrow. The specimen pictured was taken from a male not exposed to strain SF2.1(Gfp).