Fig 1.
Microsporidia MB localization in the polytrophic An. arabiensis ovariole.
(A) Schematic representation of the ovariole structure showing the germarium, the secondary follicle, and the primary follicle (with the oocyte and nurse cells in one chamber surrounded by the epithelial follicular cells). The germline stem cells (GSCs) and cystocytes are located in the germarium. (B & C) Images of a Microsporidia MB negative control ovariole obtained from a two-day-old An. arabiensis mosquito showing the localization of stem cell daughter cells, cystocytes (in blue), following cell divisions within the germarium (the blue signal represents newly synthesized DNA of cystocytes as detected by Click-iT Edu staining). (D-F) Images of a Microsporidia MB-infected An. arabiensis ovariole showing the distribution of the symbiont within the ovariole. Microsporidia MB is present in the germarium (germ) inside the GSCs (white arrowheads pointing to the GSC nucleus) and cystocytes (white arrow pointing to the cystocyte nucleus), the secondary follicle (SF), and the primary follicle (PF) i.e., the nurse cells (nc) and the oocyte (Oc). (Green represents Sytox Green DNA staining, image D, red represents Microsporidia MB-specific FISH probe staining, Image E, and a merged image of the green and red channel, Image F). Scale = 10μm. (Images are representative pictures of >100 independent observations).
Fig 2.
Localization of Microsporidia MB within the germarium and follicles across the different developmental stages of An. arabiensis ovariole.
(A) Images showing Microsporidia MB localization in the germarium, secondary, and primary follicles in all ovariole developmental stages. Microsporidia MB is spread throughout the primary follicle during the previtellogenic phase of development (stages I-III). As development progresses to the vitellogenic phase (stages III-VI), microsporidia symbiont cells accumulate in the anterior end of the primary follicle oocyte in high densities. The upper panels (a-g) represent the Sytox Green DNA channel signal, the second row of panels (a’-g’) represents Microsporidia MB-specific CY-5 channel signal and the panels in the third row (a”-g”) represent merged images of both signals. Scale = 25μm. (B) The relative area of the primary follicle oocyte occupied by Microsporidia MB symbiont as measured using the Sytox Green DNA channel signal. Microsporidia MB occupies a small area of the primary follicle oocyte in the previtellogenic stages of development and increases significantly during the vitellogenic stages of development (χ2 [5] = 57.13, p < 0.01).
Fig 3.
Microsporidia MB development in the ovariole.
(A) A typical microsporidia lifecycle showing the three microsporidian developmental stages i.e., the infective stage, proliferative, and sporogonic stages (figure adapted from [21]). (B-H) Confocal microscopy images showing the developmental stages of Microsporidia MB observed in infected An. arabiensis ovarioles. (B-D) Images of an ovariole with different sizes of Microsporidia MB cells ranging from 2μm in the germarium and secondary follicle (white arrowhead) to 5μm in the nurse cells and oocyte of the primary follicle, green and blue arrowheads, respectively. (E-G) Images showing Microsporidia MB cells in the merogony stage of proliferation in the oocyte (white arrowheads) and the presence of newly synthesized DNA in actively proliferating cells (H-J). The first column (panels B, E, and H) in green represents the Sytox Green DNA staining signal, the second column (panels C and F) in red represents the Microsporidia MB-specific CY-5 staining signal while (panel I) in blue represents Click-iT Edu staining signal and the third column (panels D, G, J) represent merged images of two channels. Scale = 10μm.
Fig 4.
Microsporidia MB infection rates in primary follicles and vertical transmission (VT) rates.
Microsporidia MB distribution in infected female ovaries was investigated and compared to vertical transmission rates. (A-B) A majority (97%) of the primary follicles within infected ovarioles had Microsporidia MB while the symbiont was completely absent in 3% of the primary follicles (B, white arrowhead). However, the germarium (germ) and secondary follicles (SF) of the 3% primary follicles that lacked Microsporidia MB were consistently infected with Microsporidia MB. Microsporidia MB was detected in 61% of sugar-fed (male & female) and 63% of blood-fed (female) adult offspring of Microsporidia MB-infected mothers. (C) Microsporidia MB was found to be present in both ovaries of infected female gonads investigated. Green represents Sytox Green general DNA staining while red represents Microsporidia MB-specific CY-5 probe staining.