Fast sequence and expression evolution of mir-977
The inference of the origination of mir-977 is based on the phylogenetic history of the entire cluster shown in Fig. 1. With three mosquito (Culicinae) genomes as outgroup (see [11]) together with all the in-group Drosophila genomes, every member of the miR- 972 cluster can be placed on the phylogenetic tree in the context of the 10 miRNA genes of the cluster. This is a cluster that has a “track record” of generating de novo miRNAs continually [10, 11] . We now stated the de novo origin of mir-977 with greater circumspection in the context of other younger de novo miRNAs.
Focusing on the mir-977 locus from the miR-972 cluster, we examined its nucleotide substitution rates among Drosophila lineages (Fig. 1a). This X-linked gene originated de novo prior to the origin of the Drosophila clade (more than 60 Myrs ago). The seed sequence in its mature product is almost identical between the melanogaster and virilis groups. However, younger Drosophila lineages show much faster evolution. Indeed, while the conservation statistic is 0.616 between D. virilis and D. melanogaster, it is 1.096 between D. simulans and D. melanogaster [11] .The 1.8 fold change is significant, (P < 0.05, Fisher’s exact test). Furthermore, this gene was altogether lost in the D. pseudoobscura lineage [11].
In addition to its fast sequence evolution, expression levels of mir-977 have also diverged among species. Using public small miRNA-seq data (see Methods and Additional file 1: Table S1), we found that mir-977 exhibits high and medium expression in D. melanogaster and D. virilis but rather low and nearly undetectable expression in D. simulans and D. erecta. The nearly undetectable expression in D. erecta might be due to multiple hairpins in its secondary structure, which would lead to abnormal miRNA processing. Furthermore, mir-977 has higher expression variance in the testes of multiple D. melanogaster lines than the conserved miRNAs that have important developmental functions (miR-184, bantam etc.; see Additional file 1: Table S2). Given its disappearance in the D. pseudoobscura and D. erecta lineages, fast evolution of mir-977 sequence and expression levels in D. melanogaster indicate its impending elimination from that genome as well.
mir-977 gene deletion
To further investigate the possible fate of mir-977 in D. melanogaster, we wanted to assay its effect on fitness. A deletion of this miRNA at its locus is necessary to perform robust experiments of this sort. While a large collection of targeted knockouts of D. melanogaster miRNAs does exist [27], the deletion that eliminates mir-977 in that set also disrupts other loci. Therefore, we set out to generate a specific knockout of mir-977 in D. melanogaster.
To achieve a complete deletion of the locus, we used the Transcription activator-like effector nuclease (TALEN) technology [28]. This approach involves designing a nuclease domain that binds next to the mir-977 mature sequence and makes double-strand breaks in the germline. Imperfect non-homologous end joining repair of these breaks results in deletions (Fig. 2a, upper panel). We used the identical w1118 white-eyed background for the wildtype and the miRNA knockout (for details, see Additional file 2: Supplementary text 1) and tried our best to avoid the possible off-target effects of TALEN (for details, see Additional file 2: Supplementary text 2). After TALEN injection, we performed a series of crosses to isolate and test candidate mutants (Fig. 2a, lower panel). We succeeded in identifying a 21 bp deletion that spans the mature mir-977 sequence (Fig. 2b). We then checked mir-977 expression in testes, the tissue where the wild-type miRNA is exclusively found. We were unable to detect any mature mir-977-5p product (Fig. 2b), while expression of the nearby miR-975 is normal. We therefore conclude that we have successfully constructed a null allele of mir-977.
mir-977’s effect on male fitness
Having established a precise mir-977 deletion in a defined genetic background, we are in a position to assay its fitness effects. Since mir-977 is expressed exclusively in testes, we focused on measuring male fitness components. The same w1118 background is used in the experiments of the mir-977 KO flies and in the wildtype control.
Male fertility
We first surveyed total male fertility, a complex phenotype that comprises multiple steps process from successful mating to production of adult offspring. We started by measuring the overall number of adult progeny produced by females of the same genotype mated to control vs mir-977− males. Strikingly, we observed a significant increase (43.6%, Student’s t test P = 0.001, N = 15) of male fertility in deletion males (Fig. 3a, left panel and Additional file 1: Table S3). It is quite unusual for a gene knockout to outperform the functional gene in such an obvious way. Although the mir-977 deletion was generated on the same genetic background as the control, it is still possible that an off-site mutation is responsible for the phenotypic effect we observe. To test this, we generated an independent five base-pair deletion in the mir-977 mature region (listed as mir-977KO-2 in Materials and Additional file 1: Figure S1a). We again found an increase in male fertility associated in mir-977 disruption (35.3%, Student’s t test P = 0.001, N = 15; Additional file 1: Figure S1b and Table S3). We thus conclude that the deletion of mir-977 itself is indeed responsible for the increase in male fertility we observe.
To further tease apart individual male fertility components (ovulation stimulation and sperm quality), we counted egg production and egg hatchability separately after mating. Deleting mir-977 increased egg production by 38.4% (Fig. 3a, right panel and Additional file 1: Table S4; Student’s t test P = 0.04, N = 12) compared to control, while slightly, and not statistically significantly, decreasing egg hatchability (Additional file 1: Table S4; 3.1%, Mann Whitney test P = 0.74, N = 12). It thus appears that the male fertility benefit of the mir-977 deficiency is entirely due to an increase in ovulation induction.
Meiotic drive
Since mir-977 is X-linked, it seems plausible that it could exert a fitness effect on the X chromosome at the expense of the Y. We therefore asked whether there is sex-linked meiotic drive associated with this new miRNA [29,30,31]. To score meiotic drive, we crossed miR977− or wild type males to w1118, mir-977+ females. We then compared the relative abundance of female and male progeny. Their ratio reflects distortion in sex chromosome transmission. We found a slight, but statistically insignificant, sex ratio distortion effect (3.9%, Student’s t test P = 0.61, N = 20) of mir-977 deletion (Fig. 3b and Additional file 1: Table S5.
Male viability
We next turned to examining the potential effect of knocking out mir-977 on male viability. We crossed mutant or wild type males to females that bore the same miRNA genotype on one sister X chromosome and a visible balancer chromosome (FM7c, see Methods) on the other. By comparing the abundance of M/Y in the progeny males (Fig. 3c, Additional file 1: Table S6), where M is either mir-977− or mir-977+, we can measure the effect of the deletion on male viability. The deficiency improved male viability significantly (7.1%, Binomial test, P = 0.012). Since mir-977 expression is confined to males, its deletion should not affect females. To test this, we counted female survival in the same experiment. We found no significant difference between control and mir-977− females (Binomial test, P = 0.48; Additional file 1: Table S6). This again suggests that the effects we observe are the direct consequence of mir-977 disruption.
Mating success
Finally, we assayed male mating ability. It has been well established [32,33,34] that in addition to success in achieving mating and inducing ovulation, males protect their sperm from competition by subsequently mating males. We already measured ovulation rates and now turn to assessing possible effects of mir-977 knockout on mating success and sperm competition.
To assay mating success, w1118 females were mated to either control or mir-977 knockout males (Stage I). After 2.5 days, these mated females were presented with reference males for a second mating (Stage II) and their mating rates at stage I and II were measured (see Methods; Fig. 3d). Our observations show that the mir-977 deletion did not affect mating success but conferred an advantage over wild type males in reducing female receptivity (43.9% vs 22.5%, Chi-square test P = 0.04, N = 40). Thus, we again see a beneficial effect of mir-977 elimination on male mating success.