Wolbachia-mediated male killing is associated with defective chromatin remodeling

Abstract

Male killing, induced by different bacterial taxa of maternally inherited microorganisms, resulting in highly distorted female-biased sex-ratios, is a common phenomenon among arthropods. Some strains of the endosymbiont bacteria Wolbachia have been shown to induce this phenotype in particular insect hosts. High altitude populations of Drosophila bifasciata infected with Wolbachia show selective male killing during embryonic development. However, since this was first reported, circa 60 years ago, the interaction between Wolbachia and its host has remained unclear. Herein we show that D. bifasciata male embryos display defective chromatin remodeling, improper chromatid segregation and chromosome bridging, as well as abnormal mitotic spindles and gradual loss of their centrosomes. These defects occur at different times in the early development of male embryos leading to death during early nuclear division cycles or large defective areas of the cellular blastoderm, culminating in abnormal embryos that die before eclosion. We propose that Wolbachia affects the development of male embryos by specifically targeting male chromatin remodeling and thus disturbing mitotic spindle assembly and chromosome behavior. These are the first observations that demonstrate fundamental aspects of the cytological mechanism of male killing and represent a solid base for further molecular studies of this phenomenon.

Figure 1. Formation of the gonomeric spindle in eggs obtained from KOS-10 Wolbachia-infected Drosophila bifasciata females.

Eggs fixed 20 minutes AED were incubated with antibodies against β-tubulin (green), acetylated-α-tubulin (blue), and counterstained with Hoechst 33258 (red). (A) metaphase of the second meiosis: two anastral spindles, separated by a large microtubule aster, are aligned in tandem orthogonal to the egg surface. (B) Pronuclear apposition: male and female pronuclei come close within a large microtubule aster nucleated by two centrosomes derived by the male-basal body inherited centriole. (C) Metaphase of the first mitosis: the gonomeric spindle is formed by two closely apposed thin spindles sharing common poles; the parental complements move independently to the metaphase plate. (D) Anaphase of the first mitosis: parental complements start to migrate synchronously to the opposite poles. (E) Early telophase: midzone microtubules form the spindle midbody and daughter nuclei have reached the opposite poles. (F) Telophase of the second mitosis: two sister spindles hold four daughter nuclei. (G,H) Metaphase-like spindles: these gonomeric spindles appear disorganized and the two spindles that hold the parental complements are distant from each other. (I) Arrested anaphase spindles: chromosomes are arrested at the metaphase plate and centrosomes detached from the poles (arrows). (J) Abnormal anaphase: chromosomes move in a disorderly fashion to the opposite poles of the spindle. (K) Abnormal telophase: the spindle lacks a distinct midzone and chromatin forms large bridges. (L) Irregular second mitosis: spindles are unusually elongated and the chromosomes are stretched among the opposite poles. Arrowheads point to remnant of the sperm tail; Pb, polar bodies. Scale bar is 10 µm.

Figure 2. Improper chromosome segregation during the intravitelline mitoses.

Black and white panels represent DNA staining alone; colour panels represent microtubules (green), DNA (red), and the sperm tail (blue). (A,A′) Detail of a normal embryo during early telophase of the seventh nuclear division cycle: sister chromatids have migrated at the opposite poles of the spindles to form daughter nuclei. Detail of embryos during early telophase (B,B′) or late anaphase (C,C′) of the seventh mitosis showing abnormal spindles: sister chromatids either do not separate and stay at the spindle equator (arrows in B) and the spindles are barrel-shaped (arrows in B′) or begin to separate, but do not migrate properly lagging in the spindle midzone or forming chromatin bridges (arrows in C), spindles do not show apparent defects at this stage (arrows in C′). (D,D′) Unfertilized eggs fixed 1 hour AED: note the anastral barrel spindles. (E,E′). Abnormal fertilized embryos fixed 1 hour AED: abnormal mitotic figures range from barrel-shaped biastral spindles (arrows in E′) to aberrant late telophase spindles in which the chromatin form dense bridges (larger arrows in E) and the centrosomes maintain their position closest to daughter nuclei (arrowheads in E′); late telophase spindles may be still recognized by the midbody remnants (small arrows in E′); double arrows in E′ point to the sperm tail. Bacteria associate with the poles of biastral spindles, but not with the poles of the anastral spindles. Scale bar is 4 µm.

Figure 3. Defects of chromatin condensation and spindle organization during the syncytial blastoderm mitoses.

Eggs fixed during the syncytial blastoderm stage and incubated with antibodies against β-tubulin (green), acetylated-α-tubulin (blue), and counterstained with Hoechst 33258 (red). (A,B) Prophase, (C,D) metaphase/anaphase, (E,F) telophase of the tenth nuclear division cycle: chromosomes have condensation defects (arrows in A, C) or do not migrate properly (arrowheads in C); telophase spindles without nuclei (arrows in E) or with only one daughter (arrowheads in E) are seen; although the poles of these spindles lack nuclei they have large asters to which bacteria are associated. (G) Detail of the anterior pole of an embryo during late telophase of the eleventh nuclear division cycle showing marked chromatin bridges (arrows). (H) Detail of an abnormal embryo showing irregular chromatin masses and free centrosomes (arrowheads). Scale bar is 4 µm.

Figure 4. Nuclear condensation defects are amplified at cellularization.

Embryos are stained for DNA (left panels) and microtubules (right panels). Cellular blastoderm is usually formed by evenly spaced nuclei at the same condensation stage and of the same size and dimensions (A) surrounded by honeycomb microtubular baskets (A′). Abnormal cellular blastoderms are characterized by small (arrow in B) or larger (arrows in C) clusters of picnotic nuclei scattered among normal looking blastoderm nuclei; the honeycomb microtubular baskets are often altered in these embryos (arrowheads in B′, C′). Scale bar is 15 µm.

Figure 5. Embryos at later stages of development have highly defective areas.

Embryos were fixed after 15–20 hours AED and stained for microtubules (green) and DNA (red). (A) Normal developing embryos in which the shortening of germ band is completed: segments are well evident. (B,C) Defective embryos after the retraction of the germ band: (B) the integrity of some segments is affected (arrows) and (C) large areas of the embryos are incompletely formed and still showed mitotic divisions (asterisks). (D) Detail of an embryo during germ band shortening: note the lack of differentiation of the anterior region of the body (arrows). Insets are details of the surface areas. Scale bar is 25 µm in the main panels and 7 µm in insets.

Figure 6. Sxl staining reveals that only male embryos have improper chromatin condensation.

Embryos were stained for DNA (larger panels) and Sxl (insets, green). Sxl positive staining is associated with embryos showing normal blastoderm (A) and proper segmentation (B). Sxl antibody fails to stain embryos that have abnormal blastoderm (C) and improper segmentation (D). Scale bar is 25 µm in the main panels and 70 µm in insets.

Figure 7. The early arrested phenotype is not due to Wolbachia.

Eggs at different times AED were fixed and incubated with antibodies against β-tubulin (green), acetylated-α-tubulin (blue), and counterstained with Hoechst 33258 (red). Unfertilized eggs showed cytoplasmic asters that increase in number with age (A,B,C, arrows); old eggs also display smaller cortical asters (arrowheads). (E) Occasionally a sperm tail is found within the eggs that show cytoplasmic asters (arrowheads). Bacteria are associated with the cytoplasmic arrays of microtubules (arrowheads, inset D). (F) Unfertilized eggs from the uninfected KOS1 females also contain cytoplasmic asters. Bacteria are also observed at the poles of the meiotic-like spindles (arrowheads, F,G), where aster-like microtubule arrays are found (arrows, F′,G′). Scale bar is 25 µm in A,B,E; 15 µm in C; 4 µm in D; 5 µm in F,G.

Figure 8. The focus of the cytoplasmic asters contains centrosomal components.

Fertilized (A,B) and unfertilized (C–F) eggs obtained by KOS10 females are incubated with antibodies against β-tubulin (green); Cnn and γ-tubulin (red) and counterstained with Hoechst 33258 (blue). Cnn and γ-tubulin are evident at the spindle poles of normal developing embryos and at the focus of the cytoplasmic asters (arrows, A–D). (E) Detail of an unfertilized oocyte at the end of meiosis with a cluster of centrosomal material within the remnant of the central aster (arrow); Pb, polar bodies. (F) Cnn aggregates are found within the cytoplasm (arrows) and around the meiotic-like spindles (F, arrows), but not at their poles (inset F, arrowheads), despite the presence of microtubule asters and associated bacteria. Bar is 25 µm in C and 4 µm in all other panels.