Identification and characterization of the doublesex gene of Nasonia

Abstract

The doublesex (dsx) gene of the parasitic wasp Nasonia vitripennis is described and characterized. Differential splicing of dsx transcripts has been shown to induce somatic sexual differentiation in Diptera and Lepidoptera, but not yet in other insect orders. Two spliceforms of Nasonia dsx mRNA are differentially expressed in males and females. In addition, in a gynandromorphic line that produces haploids (normally males) with full female phenotypes, these individuals show the female spliceform, providing the first demonstration of a direct association of dsx with somatic sex differentiation in Hymenoptera. Finally, the DNA binding (DM) domain of Nasonia dsx clusters phylogenetically with dsx from other insects, and Nasonia dsx shows microsynteny with dsx of Apis, further supporting identification of the dsx orthologue in Nasonia.

Figure 1

DM domain gene tree. The Nasonia dsx clusters with dsx of other insects. The most parsimonious tree (consensus of 20 trees with 222 steps) and a neighbor-joining tree obtained using the amino acid sequences of the DM domain region (67 total characters, 42 that are parsimony-informative) are shown with bootstrap supports above the branches. Many of the DM domain genes used in this analyses are predicted gene models.

Figure 2

Amino acid sequence alignment of the Nasonia dsx and other insects. Only the two conserved regions are presented in the figure, including the DNA binding (DM) and the dsx dimer. The zinc chelating residues in the DM domain are in bold in the reference pfam sequence(Finn et al., 2006). Putative conserved residues that distinguish the dsx DM domain from the DM domain of other proteins are shown in dotted boxes. The female isoforms were used for the dsx dimer region alignment. The predicted amino acid sequences of the 3′ region of the male isoform are shown for Nasonia and Trichomalopsis.

Figure 3

Male and female spliceforms of Nasonia dsx. RT-PCR of males, females and gynandromorph RNA. Arrows indicate male specific and female specific splice fragments. Lane 1-3: adult male; lane 4-6: adult HiCD12 haploid gynandromorph, morphologically male; lane 7-9: adult female; lane 10-11: adult HiCD12 haploid gynandromorph, morphologically female; lane 12: negative control; lane 13: 100bp Molecular marker.

Figure 4

Insect dsx gene models. The Nasonia dsx male and female spliceforms are compared to dsx of D. melanogaster and A. mellifera. See text for details.

Figure 5

An insect species trees based on the conserved dsx amino acid sequences. The most parsimonious tree (consensus of 20 trees with 335 steps) and a neighbor-joining tree obtained using the combined amino acid sequences of the DM-domain region and the dsx dimer region (138 total characters, 89 that are parsimony-informative) are shown with bootstrap support above the branches.