Sex and the single embryo: early deveiopment in the Mediterranean fruit fly, Ceratitis capitata

Abstract

Background: In embryos the maternal-to-zygotic transition (MTZ) integrates post-transcriptional regulation of maternal transcripts with transcriptional activation of the zygotic genome. Although the molecular mechanisms underlying this event are being clarified in Drosophila melanogaster, little is know about the embryogenic processes in other insect species. The recent publication of expressed sequence tags (ESTs) from embryos of the global pest species Ceratitis capitata (medfly) has enabled the investigation of embryogenesis in this species and has allowed a comparison of the embryogenic processes in these two related dipteran species, C. capitata and D. melanogaster, that shared a common ancestor 80-100 mya.

Results: Using a novel PCR-based sexing method, which takes advantage of a putative LTR retrotransposon MITE insertion on the medfly Y chromosome, the transcriptomes of individual early male and female embryos were analysed using RT-PCR. This study is focused on two crucial aspects of the onset of embryonic development: sex determination and cellular blastoderm formation. Together with the three known medfly genes (Cctransformer, Cctransformer2 and Ccdoublesex), the expression patterns of other medfly genes that are similar to the D. melanogaster sex-determination genes (sisterlessA, groucho, deadpan, Sex-lethal, female lethal d, sans fille and intersex) and four cellular blastoderm formation genes (Rho1, spaghetti squash, slow-as-molasses and serendipity-alpha) were analyzed, allowing us to sketch a preliminary outline of the embryonic process in the medfly. Furthermore, a putative homologue of the Zelda gene has been considered, which in D. melanogaster encodes a DNA-binding factor responsible for the maternal-to-zygotic transition.

Conclusions: Our novel sexing method facilitates the study of i) when the MTZ transition occurs in males and females of C. capitata, ii) when and how the maternal information of "female-development" is reprogrammed in the embryos and iii) similarities and differences in the regulation of gene expression in C. capitata and D. melanogaster. We suggest a new model for the onset of the sex determination cascade in the medfly: the maternally inherited Cctra transcripts in the female embryos are insufficient to produce enough active protein to inhibit the male mode of Cctra splicing. The slow rate of development and the inefficiency of the splicing mechanism in the pre-cellular blastoderm facilitates the male-determining factor (M) activity, which probably acts by inhibiting CcTRA protein activity.

Figure 1

Primer sequences, amplicon size and structure of the genomic region of the considered medfly sex-determination and cellularization genes. Sex-specific amplicon sizes are presented where appropriate. The genomic structure is shown only for amplicons that span more than one exon and is complete for only the Cctra and Cctra2 genes.

Figure 2

The novel molecular sexing system in Ceratitis capitata. A) Schematic representation of the Y chromosome-derived repetitive sequences. Y114 (red arrows) is the reference sequence, which was used as probe in the in situ experiments and it was the first clone derived from these sequences [14,15]. The AT-rich region (blue boxes) is a 200 bp-long sequence characteristic of Y114. Black arrows indicate CcYf and CcYr primer positions. B) Amplification patterns of genomic DNA from individual males and females using the CcYf/r primers (see Methods). C) Developmental patterns of amplification products from genomic DNA from individual male and female C. capitata. E: embryos 3, 10 and 15 hr after oviposition; L 3rd: 3^rd instar larvae; P 7 d: 7 day old pupae; A 3 d: 3 day old adults.

Figure 3

Nucleotide alignment. Nucleotide alignment of the male-specific 727 bp and 250 bp sequences and female-specific 242 bp sequence with the Y-specific repetitive sequence [GenBank:AF115330]. Black shading represents areas of identity within all four sequences; grey shading represents areas of identity between three sequences only. Underlined sequence represents the inverted terminal repeats of the MITE.

Figure 4

Male-specificity of the 727 bp sequence. A) Southern blot analysis on SspI-digested male and female genomic DNA using the male-specific 727 bp band as a probe. B) Localization of the sequence 727 bp on the Y chromosome by in situ hybridization on mitotic chromosomes.

Figure 5

RT-PCR expression pattern analysis during the early stages of embryogenesis. For each of the genes analyzed by RT-PCR the expression pattern in syncitial-stage embryos is reported together with control RT-PCR amplications on cDNA derived from unfertilized eggs and adult heads. The same primers used in RT-PCR were also used in PCR amplifications on genomic DNA. A) Expression patterns of the three known C. capitata sex determination genes: Cctra2, Cctra and Ccdsx. For the Cctra gene, the 1.1 kb splicing form is the expected male-specific mRNA, while the 0.7 kb is the female-specific form. For the Ccdsx gene the 579 bp splicing form is the expected female-specific mRNA, while the 327 bp is the male-specific form. Absence of amplification of Ccdsx from adult male genomic DNA is probably due to the presence of a long intron in the male-specific amplicon (see Figure 1). B) Expression patterns of medfly genes that share similarities to known D. melanogaster sex determination genes. C) Expression patterns of medfly genes that share similarities to known D. melanogaster cellular blastoderm formation genes. D) Expression pattern of Cczelda gene, similar to the D. melanogaster key regulatory gene of the MTZ transition event.

Figure 6

Schematic representation of Cctra transcripts detected in 6 h embryos. The colored boxes represent the exons. Male-specific exons have the prefix ms. Unspliced, spliced, and partially processed transcripts were detected in male and female embryos.

Figure 7

Timing of gene expression during embryogenesis in C. capitata and D. melanogaster. A schematic representation of the medfly and D. melanogaster embryogenesis stages, from oviposition (0 h) to hatching (48 h in the medfly, 24 h in D. melanogaster). The zygotic expression onset of crucial genes of the sex determination cascade and the two zygotic expression waves are reported in both species.