Figure 1.
Isopod ovary organisation (A) and inferred infection dynamics (B).
A: The germarium is composed of oogonia that mature and increase in diameter as they move away from the germarium in the course of ovary maturation The oocytes are encased within two sheets of follicle cells and a layer of connective tissue (dark gray). At spawning muscles compress the ovary to expel the oocytes through the oviduct, which is on the germarium side. B: We propose two non exclusive hypotheses could explain Wolbachia enrichment in the course of oocyte and ovary maturation (Immature, Mature I, Mature II stages): oocyte selection (oocytes uninfected by Wolbachia are preferentially destroyed by oosorption) and/or maturing oocytes acquire Wolbachia secondarily from somatic tissues through follicle cells.
Figure 2.
Oocyte cohorts (mean diameter ± s.e.) and their repartition in each ovary.
Depending on their maturation stage, ovaries present one to three oocyte cohorts corresponding to oocyte developmental categories called Small (S), Medium (M) and Large (L). According to these categories and the cohorts' mean diameter, we ranked the 13 ovaries along a maturation scale. The sampling broke down into three ovary maturation stages: Immature (with only S oocytes), Mature I (with S and M oocytes) and Mature II (with S, M and L oocytes).
Figure 3.
FISH detection of Wolbachia in ovaries, infected (A) versus control, uninfected (B).
The germaria are on the right borders, areas undergoing degradation on the maturing side of the ovaries (**). Oocyte nuclei are sometimes visible as dots (meiotic prophase) (*). A: The Wolbachia were present in oocytes of all sizes (red arrowheads) and their follicle cells (red arrows), though many oocytes remained uninfected (green arrowheads), especially the smaller ones. B: In uninfected ovaries. Red: Wolbachia FISH probe W1,2-Cy3, green: phalloidin, blue: DAPI.
Figure 4.
Main effect plots of Wolbachia infected oocyte proportion in the course of ovary maturation and oocyte development.
The Y axis is labelled on the logit scale of the predictor (i.e. the probability scale of the response). The multinomial logistic regression showed that the proportion of Wolbachia infected oocytes per ovary depended on both the oocyte cohorts (P<2.2e−16) and the ovary maturation stage (P<2.2e−16). The proportion of infected oocytes remains stable between Immature and Mature I ovaries but increases between Mature I and Mature II ovaries. The proportion of infected oocytes increases throughout oocyte development.
Figure 5.
Close-up on FISH detection of Wolbachia in infected ovaries.
Wolbachia appear in red and mostly cluster around the nucleus (blue). The yellow patterns at the periphery of some oocytes correspond to a type of vesicle which content autofluoresces both in red and green. In this Mature I ovary the germarium is on the left side, then some Small uninfected oocytes (e.g. *), and Medium size oocytes with representatives from the category “poorly infected” (**) and highly infected (***). Red: Wolbachia FISH probe W1,2-Cy3, green: phalloidin, blue: DAPI.
Figure 6.
Average number of uninfected and infected (poorly and highly infected) oocytes per ovary for each ovary maturation stage (A) and in each oocyte developmental category (B).
As ovaries mature (A), the total number of oocytes increases between the first two maturation stages and decreases between Mature I and Mature II. The numbers of uninfected and poorly infected oocytes follow the same pattern, whereas in contrast, the number of infected oocytes per ovary rises throughout ovary maturation. Again, as oocytes develop (B), the numbers of uninfected and poorly infected oocytes decrease while the number of infected oocytes increases between S and M categories only. Oocyte numberings were recorded from (A) one Immature ovary, six Mature I and six Mature II ovaries, (B) 13 ovaries containing S oocytes (all maturation stages), 12 ovaries containing M oocytes (Mature I and Mature II ovaries), and six ovaries containing L oocytes (Mature II ovaries). Standard errors are presented in the main text.
Figure 7.
Average intensity projections of ovaries: Wolbachia (red and grayscale) detection in infected ovaries at different maturation stages: Immature (A), Mature I (B 1 and 2), Mature II (C), versus control uninfected ovary (D).
The germaria are at the bottom, the scale bars placed in front of the oviduct insertion. A: Oocytes are only present along the thin band of the germarium (30.5% infected; infected oocytes: e.g. red arrowheads; uninfected ones: e.g. green arrowheads). B: Wolbachia are present as a mass near the nucleus of oocytes of all sizes (e.g. red arrowheads) and their follicle cells (infection there appears as a network surrounding the oocytes, e.g. red arrows), though many oocytes remained uncolonized (e.g. green arrowheads), especially the smaller ones. In the B1 ovary the colonized oocytes are roughly arranged as triangles, one tip stemming from the germarium side and expanding to the mature side (51% of infection), while the pattern in B2 (67% of infection) is more representative of this category, in so far as the colonization is not so linear. Also some colonized oocytes are not surrounded by infected follicle cells (e.g. red circles) and vice versa (e.g. red squares). C: Mature II is the most advanced stage in maturation (infected oocyte: e.g. red arrowhead; uninfected oocyte: e.g. green arrowhead). In addition to the Wolbachia signal the inside of the oocytes is filled with autofluorescing material, probably vitellogenin. D: Uninfected ovary. Red and grayscale: Wolbachia FISH probe W1,2-Cy3, green: phalloidin, blue: DAPI.
Figure 8.
Wolbachia co-detection with DAPI and FISH in infected (A, B) versus control, uninfected ovaries (C, D).
Wolbachia appear in purple (e.g. purple arrowheads). The DAPI staining (blue or grayscale) of oocyte nuclei (e.g. *) and Wolbachia is lower than the fluorescence emitted by follicle cells nuclei (e.g. blue arrowheads): its observation requires overexposing the nuclei of the follicle cells (e.g. blue arrows). In infected females (A, B) Wolbachia FISH and DAPI signals are detected around the nuclei in infected oocytes (e.g. purple arrowheads) while in uninfected oocytes only the nuclei are labelled (white arrowheads). In uninfected females only background was observed with the FISH probe (C: e.g. yellow arrowheads) which did not co-localize with DAPI staining (D: e.g. yellow arrow). Red: Wolbachia FISH probe W1,2-Cy3, blue and grayscale: DAPI.