Development ofPhasmarhabditis hermaphrodita(and members of thePhasmarhabditisgenus) as new genetic model nematodes to study the genetic basis of parasitism

Abstract

The genetic mechanisms of how free-living nematodes evolved into parasites are unknown. Current genetic model nematodes (e.g. Caenorhabditis elegans) are not well suited to provide the answer, and mammalian parasites are expensive and logistically difficult to maintain. Here we propose the terrestrial gastropod parasite Phasmarhabditis hermaphrodita as a new alternative to study the evolution of parasitism, and outline the methodology of how to keep P. hermaphrodita in the lab for genetic experiments. We show that P. hermaphrodita (and several other Phasmarhabditis species) are easy to isolate and identify from slugs and snails from around the UK. We outline how to make isogenic lines using 'semi-natural' conditions to reduce in-lab evolution, and how to optimize growth using nematode growth media (NGM) agar and naturally isolated bacteria. We show that P. hermaphrodita is amenable to forward genetics and that unc and sma mutants can be generated using formaldehyde mutagenesis. We also detail the procedures needed to carry out genetic crosses. Furthermore, we show natural variation within our Phasmarhabditis collection, with isolates displaying differences in survival when exposed to high temperatures and pH, which facilitates micro and macro evolutionary studies. In summary, we believe that this genetically amenable parasite that shares many attributes with C. elegans as well as being in Clade 5, which contains many animal, plant and arthropod parasites, could be an excellent model to understand the genetic basis of parasitism in the Nematoda.

Gastropod parasitic nematodes (Phasmarhabditis sp.) are attracted to hyaluronic acid in snail mucus by cGMP signalling

“…Wales (Andrus & Rae, 2018). Our strain was isolated from a snail (Oxychilus draparnaudi) collected from Pembrokeshire, Wales (Andrus & Rae, 2018). Research into P. californica has concentrated on its recent description (Tandingan de Ley et al…”

“…Wild strains of Phasmarhabditis were sub-cultured by growing them in White traps (described in Andrus & Rae, 2018) where approximately 100 nematodes were added to a rotting piece of Limax flavus and left for 28 days until they grew to the dauer stage and then used in experiments. For each snail species three replicate plates were used and the experiment was repeated three times.…”

“…There is no information about how wild strains of P. hermaphrodita and other Phasmarhabditis species respond to gastropod cues such as mucus. Therefore, we utilised a collection of recently isolated wild strains of P. hermaphrodita and Phasmarhabditis species (including P. californica and P. neopapillosa) (Andrus & Rae, 2018) to examine their chemoattraction behaviour to snail mucus to see if it differed from the commercial isolate.…”

Caenorhabditis elegansnematodes are not attracted to the terrestrial isopodPorcellio scaber

“…For example, phoretic associations between Pristionchus pacificus and scarab beetles (Herrmann et al, 2006), Caenorhabditis japonica and the shield bug Parastrachia japonensis (Kiontke et al, 2002;Tanaka et al, 2010), and the facultative parasite Phasmarhabditis hermaphrodita and slugs of the genus Arion or Deroceras as well as the snail Helix aspersa (Rae et al, 2009;Félix et al, 2018;Andrus & Rae, 2019). Previous studies have shown that mucus of the slug Arion subfuscus and the snail Helix aspersa act as strong chemoattractants for P. hermaphrodita (Rae et al, 2009;Andrus & Rae, 2019). This association with slugs and snails has also been observed with C. elegans, where nematodes have been recovered from the intestines and feces of Arion sp.…”

My favourite nematode – Phasmarhabditis hermaphrodita

“…Imagine a world where the discoveries of RNAi, the genetic regulation of apoptosis and the development of GFP (green fluorescent protein) that resulted in 3 Nobel prizes were due to studying the slug parasite P. hermaphrodita rather than C. elegans! Recently, P. hermaphrodita has been proposed as a model system of its own -specifically to study the evolution of parasitism (Wilson et al, 2015;Rae, 2017a;Andrus and Rae, 2018). Several attributes make it a perfect system and recently protocols have been developed that show how to culture it under lab conditions, mutagenize, mate and make isogenic lines of P. hermaphrodita just like C. elegans (Andrus and Rae, 2018).…”