Figure 1.
Schematic representation of the first embryonic cleavages in secernentean nematodes, and growth of Brugia malayi embryos in utero.
(A) Cell linage adapted from [31], [33]. Anterior daughters are to the left, posterior ones to the right. The main derivatives are indicated by dotted lines. (B) Uterus filled with early cleavage embryos. Note the variety of embryo sizes (small and large arrows). (C) Three developmental stages in one merge of confocal stacks (lower left to upper right): embryos at approximately the 12-cell, 100-cell stage prior to morphogenesis, and mature pretzel stage prior to hatching. (D) Length of the B. malayi embryo eggshell according to the embryonic stage. In each case, the longest axis of the eggshell was measured. Each data point represents one measurement.
Figure 2.
Wolbachia concentrate to the posterior pole of the egg and preferentially segregate to the P1 blastomere, to later enrich the C blastomere and subsequently colonize posterior hypodermal cells.
Merges of confocal stacks of B. malayi eggs and embryos stained for DNA (propidium iodide, red), Wolbachia (anti-WSP, blue), and cortical actin (green). (A), completion of meiosis, metaphase I, and (B) completion of metaphase, meiosis II as revealed by the presence of a polar body (PB, white arrow). White arrowheads indicate the pseudocleavage. Maternal chromatin to the anterior pole (left) and paternal chromatin to the posterior (Sp., sperm). (C) Pronuclei migration and (D) Pronuclei apposition while the three polar bodies (PBs) are extruded at the anterior pole. Note that the PBs usually lost at the 4 or 6-cell stage, can detach earlier, probably due to the fixation technique (i.e. (E)). (F) Division of P0 into the anterior AB and the posterior P1 blastomeres. (G) 3-cell stage, AB divides prior P1. (H) 4-cell stage, (I) 6-cell stage, and a (J) 12-cell stage embryo. Partial projections highlighting the dorsal (JI) and ventral (JII) blastomeres. Solid lines connect sister blastomeres on the corresponding schematic drawings. (KI to KIV) Merges of confocal stacks of a comma-stage embryo, from the surface to half depth, anterior to the left. White arrowheads and arrows highlight dorsal and ventral hypodermal cells respectively, and dots indicate lateral hypodermis. Green arrowheads highlight ventral (KII) and dorsal (KIII) muscles. Green, white, and red ovals encompass the neuroblasts, the pharynx, and the gut primordium respectively. The red arrowhead indicates the rectum (KIII, KIV). Scale bar = 6 µm.
Figure 3.
Wolbachia are not always detected in the germline precursors.
(A) to (C) Merges of confocal stacks of a 1,5 fold stage embryo (when the tail reaches half of the length of the embryo) stained for DNA (propidium iodide, red) and Wolbachia (anti-WSP, blue). Infected putative primordial germ cells Z2/Z3 are indicated by an arrowhead. (D) to (L) Merges of confocal stacks of B. malayi embryos in gastrulation stained for DNA (PI, red) and with anti-H3K4me2 (green) at the level of the putative P4 blastomere (D,E), or putative Z2 and Z3 (F). (D) and (E) are the earliest stages where the H3meK4-negative P4 blastomere was detected. (G to L) are confocal stacks in cross sections focusing on Z2 or Z3. (D) Non-infected P4; (E) Infected P4 (arrowheads in inset point to Wolbachia); (G) Non-infected Z2/Z3; (G to I) Non-infected Z2 or Z3 cell (arrow); (J to L) A Wolbachia-infected Z2 or Z3 cell (the arrow points to the cell nucleus and the arrowhead to a cluster of bacteria. Scale bar = 10 µm.
Figure 4.
(A) Overview of the adult anatomy of fixed and DAPI-stained Brugia malayi female and male. (B to F) Anatomical details of the Brugia malayi female oriented anterior top to posterior bottom. (B) Higher magnification of anterior female reproductive apparatus. A zone of dense somatic nuclei is surrounded by muscles, which have fewer nuclei. (C) Zone of hatching in one of the two uteri. Fully developed pretzel-stage larvae still in their eggshell are at the bottom. Hatched microfilariae that spontaneously align along the antero-posterior are at the top. (D) Developing embryos in the uterus. The perfectly aligned hypodermal nuclei of a lateral chord are visible. (E) Distal-most part of the uterus where sperm has concentrated, brightly stained with DAPI (i.e. in between dotted arrows, also in bright patches in (A) between D and E). (F) Posterior part of the female, showing the two ovaries running back and forth (“ovary 1”, arrows indicating the 180° turns -1, 2 and 3-, idem for “ovary 2”). The amount of sperm, embryos and microfilariae are variable among females.
Figure 5.
Wolbachia concentrate in the hypodermal lateral chords.
DNA (propidium iodide, red), and actin (green) stainings of female (A, B) and male (C, D) adult lateral chords. (E, F) The upper but not the lower chord is infected. (G,H) Both chords are uninfected. (H) The phalloidin staining reveals the gonad contractile sheath cells. (I, J) The upper lateral chord is being invaded by Wolbachia, revealed by propidium iodide. The uterus is embedded in the chord. (K, L) Surface view of a chord and (K′, L′) 90° projections showing the circumferentially oriented actin bundles in the apical part, while the Wolbachia, surround the two rows of hypodermal nuclei (see Material and Methods for technical details) in the basal part, separated by the secretory-excretory canal (arrowheads). Scale bars = 100 µm.
Figure 6.
Detection of Wolbachia by propidium iodide in the lumen of the secretory-excretory canal.
(A) One canal reaching the pore near the mouth, stained with phalloidin without fixation. (B) to (G) Confocal merges at the level of the lumen in fixed females stained for DNA (propidium iodide, red), and cortical actin (green). (B) to (D) At the level of an infected or (E) to (G) non-infected chords. (E) to (G) are close to the pore. m., muscles, arrowheads point to the lumen of the canal. (H) to (M) Live female incubated with resorufin (red) and syto11(green). (H) to (J) one focal plan in the largest section of the canal, (K) to (M) total projection of the canal, (K′) to (M′) 90° projections showing a possible excretion of Wolbachia revealed by the propidium iodide (white arrowheads). Note that the canal appears enlarged, as a consequence of the use of Sodium Azide to immobilize the worm (white bars).
Figure 7.
Wolbachia localization and segregation patterns followed during early embryogenesis.
(A) Schematic drawing of Wolbachia localization in a cross section of a Brugia malayi adult female. The Wolbachia (purple dots) are present in the cell bodies of the hypodermal lateral chords (in red), secretory-excretory canal, in the germline and in embryos. They are absent from the somatic gonad, the muscles and the intestine. (B) Model of likely segregation patterns followed by Wolbachia during early embryogenesis to colonize adult tissues. Observation of fixed embryos and adult tissues indicate that Wolbachia must follow the germline precursors in females and the C lineage leading to hypodermal cells (green solid lines). In contrast, some blastomere derivatives are not favorable to Wolbachia proliferation (i.e. blue dotted lines). In male embryos, the Wolbachia may not be maintained in the germline precursors.