Figure 1.
Meiosis genes are expressed in asexual aphids.
A. Cyclical parthenogenesis among aphidomorph insects evolved from sexual ancestors as shown in this phylogeny (adapted from [3]). Time scale on bottom is in millions of years ago (MYA). B. Structures of an asexual ovariole (top) and a sexual ovariole (bottom) are shown. Ovarioles are oriented with anterior on the left. Scale bars are shown. C. Both sexual and asexual ovaries express meiosis genes. PCR products from cDNA template (+), control template lacking reverse transcriptase (RT) during cDNA synthesis (-), or from no template (NT) are shown. D. PCR of meiosis genes from the Tucson obligately asexual pea aphid strain. Lanes contain products from PCR using Tucson ovary cDNA template (+RT), template from cDNA synthesis reactions lacking RT (-RT), and genomic DNA (gDNA). Primers are the same as those used for C.
Figure 2.
Spo11 is spliced less in asexual aphids than in sexual aphids.
A. Pea aphid Spo11 gene structure is shown. Boxes refer to exons and lines refer to introns, with bp lengths indicated inside or below, respectively. The third intron includes a 1720 bp SINE2 retrotransposon, indicated by the arrowhead. PCR primer sets used in reactions shown in Panels B, C and D are indicated by a numbered dashed line and pair of arrows. The scale bar indicates 100 bp. B. Unspliced Spo11 is detected in asexual aphids. Shown are PCRs from: genomic DNA (g); cDNA pools synthesized from RNA from sexual (“Sex”) LSR1.G1.AC ovaries, asexual (“Asex”) LSR1.G1.AC ovaries (+) and Tucson (“TUC”) pea aphid ovaries; and the RT-absent control template (-). Upper panel shows PCRs products from the second and third exons of Spo11. The bottom panel shows PCR products of the last four (left) or last six (right) exons of Spo11. DNA size standards are indicated on the marker (M) lanes of these gels. C. Spo11 intron RNA is more abundant in asexually reproducing aphids than in sexual aphids. Shown are PCRs from: genomic DNA (g); cDNA pools synthesized from RNA from Tucson ovaries, asexual or sexual LSR1.G1.AC ovaries (+); or the RT-absent control template (-). Upper panel shows PCRs products from the fourth intron of Spo11. The bottom panel shows PCR products of full-length Hop2 using the same template amounts. D. Spo11 is expressed in asexual embryos in utero. Top panel shows Spo11 PCR products using primer set 2, and the bottom panel shows Pms1 PCR product. Pools of cDNA (+) or control template from cDNA synthesis reactions lacking RT (-) were created from the tissue sources listed above the lanes.
Figure 3.
Meiosis genes are expressed in germaria and oocytes of both sexual and asexual aphids.
A. Spo11 staining. B. Hop2 staining. C. Mnd1 staining. D. Rad21 staining. Black brackets indicate the germaria. Red brackets indicate the oocyte. Black arrows point to trophocyte nuclei in the germarium. Red arrows point to follicular epithelial cells. Black arrowheads point to the oocyte nucleus when in the plane of focus. Stages of asexual embryos in focus are labeled as described [14]. When two asexual embryos are observed in the same focal plane, both stages are indicated. Hybridizations using labeled sense probe for Spo11 and for Hop2 are shown. Black scale bars refer to 20 µm (asexuals) or 100 µm (sexuals).
Figure 4.
Argonaute-family genes are differentially expressed between aphid morphs.
Shown are PCRs of aphid Argonaute-family genes Piwi3, Piwi8 and Ago3a, along with GAPDH as a loading control. Products were amplified from cDNA pools synthesized with (+) or without (-) RT from RNA from asexual or sexual LSR1.G1.AC ovaries.