Experimental tests of sex allocation theory in a simultaneous hermaphrodite using phenotypic engineering

Sex allocation theory can provide successful predictions about how individuals allocate resources into male and female reproduction. However, in simultaneous hermaphrodites, experimental support for some fundamental aspects of sex allocation theory are, so far, limited, and many studies are conducted as correlational analyses without experimental trait manipulation. This limitation is especially true in animals, where gonads are usually internal, making it difficult to experimentally manipulate the traits of a specific sex function. Therefore in my PhD project I established and performed phenotypic engineering in the free-living simultaneous hermaphroditic flatworm Macrostomum lignano, and experimentally tested two fundamental aspects of sex allocation theory, namely a saturating male fitness gain curve and the existence of a trade-off between male and female allocation. First, in order to identify candidate genes that could be involved in sex allocation, I searched sex-specific genes based on existing gene annotations from other organisms using available gene sequence databases in M. lignano. Next, I examined gene expression patterns of such candidate genes using in situ hybridization. After confirming sex specificity, I knocked down gene expression using RNA interference (RNAi), enabling me to obtain a number of male-sterile phenotypes. In Chapter II, I report one of the identified testis-specific genes, named melav2, as an example of how to confirm sex specificity, an important aim for specific manipulation of one sex function in a simultaneous hermaphrodite. Having identified male-specific genes, I next investigated how the manipulation of sperm production using one of these genes impacts on male fitness. In sex allocation theory the shape of the fitness gain curve has an important role to predict optimal sex allocation. In many simultaneous hermaphrodites saturating male fitness gain curves are predicted, but studies experimentally testing this prediction are scarce. Therefore in Chapter III, I quantitatively manipulated sperm production using dose-dependent testis-specific RNAi knock-down of the macbol1 gene, and the results provide clear experimental evidence of a saturating male fitness gain curve in M. lignano, to my knowledge the first such evidence in copulating simultaneous hermaphrodites. Another fundamental but to date poorly supported assumption of sex allocation theory is the existence of a trade-off between male and female resource allocation. In theoretical models individuals are assumed to have a limited amount of reproductive resources, and an increase in allocation in one sex function necessarily leads to a decrease in the other sex function, imposing some constraints on the individual’s sex allocation decisions. In Chapter IV, in support of this assumption, I show that experimentally hindering spermatogenesis via testis-specific RNAi knock-down of the mac-C3H-zfn gene results in an increase in ovary size, as assumed by theory. In conclusion, my thesis provides experimental evidence for two fundamental aspects of sex allocation theory, namely a saturating male fitness gain curve and the existence of a trade-off between male and female allocation in a copulating simultaneous hermaphrodite. These two findings confirm an important theoretical prediction, namely that selection favors lower male allocation when there are diminishing male fitness returns and that freed reproductive resources can flow from the male to the female function