Molecular characterization and evolution of a gene family encoding both female- and male-specific reproductive proteins in Drosophila

Abstract

Gene duplication is an important mechanism for the evolution of new reproductive proteins. However, in most cases, each resulting paralog continues to function within the same sex. To investigate the possibility that seminal fluid proteins arise through duplicates of female reproductive genes that become "co-opted" by males, we screened female reproductive genes in Drosophila melanogaster for cases of duplication in which one of the resulting paralogs produces a protein in males that is transferred to females during mating. We identified a set of three tandemly duplicated genes that encode secreted serine-type endopeptidase homologs, two of which are expressed primarily in the female reproductive tract (RT), whereas the third is expressed specifically in the male RT and encodes a seminal fluid protein. Evolutionary and gene expression analyses across Drosophila species suggest that this family arose from a single-copy gene that was female-specific; after duplication, one paralog evolved male-specific expression. Functional tests of knockdowns of each gene in D. melanogaster show that one female-expressed gene is essential for full fecundity, and both female-expressed genes contribute singly or in combination to a female's propensity to remate. In contrast, knockdown of the male-expressed paralog had no significant effect on female fecundity or remating. These data are consistent with a model in which members of this gene family exert effects on females by acting on a common, female-expressed target. After duplication and male co-option of one paralog, the evolution of the interacting proteins could have resulted in differential strengths or effects of each paralog.

Keywords: Drosophila; gene duplication; protease; seminal proteins; sex-specific expression; spermathecal proteins.

Fig. 1.

Chromosomal locations and gene order of CG9897, CG32833, and CG32834 and their orthologs in Drosophila species. In D. simulans, GD15206 is found in an unassembled part of chromosome (chr) 2R (indicated by the asterisk), whereas UN32833 and UN9897 represent unannotated copies whose sequences we determined by sequencing or BLAST. The Dsim\UN32833 sequence is only partially determined (the 142 codons at the start of the coding sequence). Color indicates gene expression pattern: pink is female-specific or female-biased; bright blue is male specific; light blue is expressed only at a low level in males; green is expressed approximately equally in both sexes. Gene order and conserved expression patterns were consistent with calls of orthology and with phylogenetic clustering (see fig. 4).

Fig. 2.

Whole animal expression patterns of Drosophila melanogaster CG32834, CG32833, and CG9897 and their orthologs in other congeners. F: female; M: male; g: genomic; N: negative control (water used as template). RpL32 is a ribosomal protein-encoding gene used as a control; primers for this gene were designed to span an intron to check for contamination of cDNA with genomic DNA. Gene names of the orthologs from conspecific species are given in figure 1. Note: (a) GF11310 is a D. ananassae homolog to the gene family but is not definitively orthologous with any one gene. GA25104 is a D. pseudoobscura homolog to the gene family but is not definitively orthologous with any one gene.

Fig. 3.

Tissue-specific expression patterns of Drosophila melanogaster CG32834, CG32833, and CG9897 and their orthologs in D. ananassae. Expression patterns are also shown for GA25104, the single D. pseudoobscura member to the gene family. L: ladder; B: body without RT; G: gonads; R: RT without gonads; g: genomic DNA; and N: negative control (water used as template). RpL32 expression was used as a control. Notes: (a) At 35 cycles, a light band appears in the male gonads and RTs without gonads of Dmel for CG9897. (b) GF11310 is a D. ananassae homolog to the gene family but is not definitively orthologous with any one gene. GA25104 is a D. pseudoobscura homolog to the gene family but is not definitively orthologous with any one gene.

Fig. 4.

Maximum-likelihood phylogeny of protein sequences for each member of the gene family. Bootstrap support based on 100 replicates is shown in italics at each node. Tip labels indicate protein names; the first three letters indicate the Drosophila species (mel: melanogaster; sim: simulans; sec: sechellia; yak: yakuba; ere: erecta; ana: ananassae; pse: pseudoobscura), and the following characters indicate the FlyBase gene name. “UN” in the gene name indicates a previously unannotated copy of the gene in D. simulans. Scale bar indicates the number of substitutions per site. Calls of orthology are consistent with phylogenetic clustering and gene order (see fig. 1): the six genes shown at the top of the figure (GD15206–GF11314) are one set of orthologs, GF11312–GG20080 are another set, and GG20079–GF11310 are the third set. The tree is rooted on the single D. pseudoobscura copy of this gene family, GA25104. Gene expression patterns from figure 2 are indicated in italicized text.

Fig. 5.

Probability of remating by Drosophila melanogaster females after initial matings between females and males knocked down for various combinations of CG32834, CG9897, and CG32833. (A) Remating probabilities at 1, 4, and 10 days after an initial mating for Trial 1. Trial 1 includes all eight possible combinations of gene knockdown for female genes CG32834 and CG9897 and male gene CG32833. Gene presence is indicated on the x-axis of each graph with a “+,” whereas knockdown is indicated by a “0.” Day 1: N = 17–37 females per treatment; Day 4: N = 15–37 females per treatment; Day 10: N = 73–88 females per treatment (three replicates combined). (B) Remating probabilities at 1, 4, and 10 days after an initial mating for Trial 2. Trial 2 included all four possible combinations of gene knockdown for female genes CG32834 and CG9897. All males used in this trial were the wild-type Canton S stock. Day 1: N = 37–45 females per treatment (two replicates combined); Day 4: N = 35–53 females per treatment (two replicates combined); Day 10: N = 88–108 females per treatment (four replicates combined).

Fig. 6.

Egg laying by Drosophila melanogaster females resulting from matings between females and males knocked down for various combinations of CG32834, CG9897, and CG32833. (A) Trial 1 used females knocked down for CG32834, CG9897, both genes, or neither gene, and males knocked down for CG32833 or not. (N = 32–58 females per treatment; total eggs laid for 5 days after mating.) (B) Trial 2 used females knocked down for CG32834, CG9897, both genes, or neither gene, mated to wild-type Canton S males. (N = 15–40 females per treatment; total eggs laid for 4 days after mating). Gene presence is indicated on the x-axis of each graph with a “+,” whereas knockdown is indicated by a “0.” Error bars indicate one standard error of the mean.