Two Simple Methods for the Collection of Individual Life Stages of Reniform Nematode, Rotylenchulus reniformis

Abstract

The sedentary semi-endoparasitic nematode Rotylenchulus reniformis, the reniform nematode, is a serious pest of cottonand soybean in the United States. In recent years, interest in the molecular biology of the interaction between R. reniformis and itsplant hosts has increased; however, the unusual life cycle of R. reniformis presents a unique set of challenges to researchers who wish tostudy the developmental expression of a particular nematode gene or evaluate life stage–specific effects of a specific treatment suchas RNA-interference or a potential nematicide. In this report, we describe a simple method to collect R. reniformis juvenile andvermiform adult life stages under in vitro conditions and a second method to collect viable parasitic sedentary females from hostplant roots. Rotylenchulus reniformis eggs were hatched over a Baermann funnel and the resultant second-stage juveniles incubated inpetri plates containing sterile water at 308C. Nematode development was monitored through the appearance of fourth-stage juvenilesand specific time-points at which each developmental stage predominated were determined. Viable parasitic sedentary females werecollected from infected roots using a second method that combined blending, sieving, and sucrose flotation. Rotylenchulus reniformislife stages collected with these methods can be used for nucleic acid or protein extraction or other experimental purposes that relyon life stage–specific data.