Infective Larvae Production and Development in Strongyloides and its role in the dauer hypothesis

Abstract

The dauer hypothesis for the evolution of parasitism in nematodes, states that dauer larvae, a specialised third larval stage present in many free living nematodes, served as a pre adaptation to parasitism and evolved into infective larvae (L3i). Unique biological features like alternating free-living sexual and parasitic parthenogenetic generations make Strongyloides spp., which includes the human pathogen S. stercoralis, an attractive model to study parasitism and the evolution thereof. One of the most striking features is that all L3is are female, although they can be produced from sexual reproduction. The main aims of this thesis were to test the dauer hypothesis for the specific example of Strongyloididae and to understand how female only progeny are formed from sexual reproduction. While Strongyloides ratti is an attractive model species as it can be maintained within its natural host, its free living stages can only be extracted from fecal cultures and many tools that are standard in model organisms are not yet available, making experimentation difficult. Therefore, I firstly set out to increase the toolkit within S. ratti. I strongly improved the protocol to culture S. ratti on bacteria seeded agar plates, which led to highly increased fecundity and survival, allowing easier experimentation. Further I devised the first working procedures for RNAi in any species of Strongyloides. As S. ratti appears refractive to microinjection, I developed an RNAi by soaking protocol which works across multiple life stages and has minimal off-target effects. Using the aforementioned RNAi technique, I studied daf-12 natively within S. ratti (the first study of its kind within Strongyloides). By suppressing DAF-12 I found that many functions of this nuclear hormone receptor are conserved between C. elegans and S. ratti. In particular, daf-12(RNAi) inhibited the formation of infective larvae as it does for dauer larvae in C. elegans. This demonstrates that the molecular mechanisms underlying dauer formation and L3i formation are conserved between species as phylogenetically distant as C. elegans and S. ratti , suggesting that DAF-12 it is a promising target for novel antihelminthics and providing support for the dauer hypothesis. In order to study the transition from free-living to parasitic, we need well-studied closely related free-living species to parasites of interest, of which we are currently lacking. After noticing that Rhabditophanes KR3021 (the closest known free living species to Strongyloides) produces dauer larvae and arrested J2 larvae (both previously unreported) under starvation conditions, I aimed to characterise this species and provide clearly lacking basic information. In addition to providing a detailed description of life cycle and morphology, I showed that the daf-12 pathway is also involved in dauer (but not arrested J2) formation making Rhabditophanes/Strongyloides a highly attractive system for the further evaluation of the dauer hypothesis. Next, by characterising spermatogenesis in S. ratti, S. papillosus and P. trichosuri (which does make males), I found that there is an uneven division resulting in viable X-bearing and non-viable nullo-X sperm. I showed that spermatogenesis itself is highly conserved within the Strongyloididae, from the presence of giant nuclei in the distal gonad, to expression and production of major sperm protein.