From Mosquito Ovaries to Ecdysone; from Ecdysone to Wolbachia: One Woman's Career in Insect Biology

Abstract

In anautogenous mosquitoes, synchronous development of terminal ovarian follicles after a blood meal provides an important model for studies on insect reproduction. Removal and implantation of ovaries, in vitro culture of dissected tissues and immunological assays for vitellogenin synthesis by the fat body showed that the Aedes aegypti (L.) (Diptera, Culicidae) mosquito ovary produces a factor essential for egg production. The discovery that the ovarian factor was the insect steroid hormone, ecdysone, provided a model for co-option of the larval hormones as reproductive hormones in adult insects. In later work on cultured mosquito cells, ecdysone was shown to arrest the cell cycle, resulting in an accumulation of diploid cells in G1, prior to initiation of DNA synthesis. Some mosquito species, such as Culex pipiens L. (Diptera, Culicidae), harbor the obligate intracellular bacterium, Wolbachia pipientis Hertig (Rickettsiales, Anaplasmataceae), in their reproductive tissues. When maintained in mosquito cell lines, Wolbachia abundance increases in ecdysone-arrested cells. This observation facilitated the recovery of high levels of Wolbachia from cultured cells for microinjection and genetic manipulation. In female Culex pipiens, it will be of interest to explore how hormonal cues that support initiation and progression of the vitellogenic cycle influence Wolbachia replication and transmission to subsequent generations via infected eggs.

Keywords: cell cycle; cell line; flow cytometry; insect reproduction; mosquito; obligate intracellular bacterium; organ culture.

Figure 1

Emergence of Hyalophora cecropia moths from 1 June through 15 July 1965 in Pawcatuck, CT, USA. Open bars, males; closed bars, females.

Figure 2

Ecdysone increases Wolbachia abundance in Aa23 cells. Panels (a,b) show profiles of Aa23 cells in the absence of ecdysone (20E) on day 5 and day 12 after plating cells at near confluence. Panels (c,d) show replicate plates with 2 × 10⁻⁶ M ecdysone added on day 0. Note that fluorescence corresponding to Wolbachia is represented by WP; fluorescence due to host cell nuclei is shifted to the far right (CP). Events on the Y-axis in panels (a,c) are similar, suggesting little effect of ecdysone during the first 5 days of exposure. On day 12, the WP has substantially increased in panel (d) relative to (b), reflecting improved yield of Wolbachia, and the CP has dropped, reflecting the negative effect of ecdysone on cell proliferation over time. Note that the CP profile in (d) shows a higher proportion of cells in G1 than in G2, as is the case with C7-10 cells [35]. This experiment was replicated with three independent biological samples.

Figure 3

Cytometric analysis of crude Wolbachia preparations from Aa23 cells directly resuspended in culture medium. Panel (a), scatter plot showing events in Q1 that interfere with quantitation of the WP in the corresponding histogram shown in Panel (b). The vertical white open arrow indicates corresponding positions in the two panels. WP, Wolbachia peak; CP, cell peak. Panel (c), an independent preparation of Aa23 cells assayed after two weeks in the absence of a C7-10 feeder layer; Panel (d), a replicate Aa23 inoculum added to a confluent monolayer of mitomycin C-treated C7-10 cells. Events in polygon T, which excludes Q1 events (shown in (a,c,d)) were used to estimate relative Wolbachia abundance.

Figure 4

wAlbB harvested from C7-10 cells treated with mitomycin C. Panel (a) shows an image of cells represented in Figure 3d after a two-week incubation with mitomycin C-treated feeder cells. Based on size (approximately 10-micron diameter) and appearance of nuclei, cells shown in the rectangle are Aa23 cells introduced as the inoculum. White arrows show nuclei of live (green) and dead (red) Aa23 cells. The oval region shows the aberrant morphology and high levels of Wolbachia (barely visible, small green dots such as those in the syncytium-like structure to the left of a white, leftward pointing arrow) that develop in the feeder layer. The black size marker that crosses the oval at left indicates 50 microns. Panel (b) shows the final wAlbB scatter plot after purification by filtration and centrifugation as described previously for C/wStri1 cells [103]. The inset shows the WP profile.