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

摘要

The sedentary semi-endoparasitic nematode Rotylenchulus reniformis, the reniform nematode, is a serious pest of cotton and soybean in the United States. In recent years, interest in the molecular biology of the interaction between R. reniformis and its planthostshasincreased;however,theunusuallifecycleof R. reniformis presentsa uniquesetofchallengesto researcherswhowishto study the developmental expression of a particular nematode gene or evaluate life stage–speci?c effects of a speci?c treatment such as RNA-interference or a potential nematicide. In this report, we describe a simple method to collect R. reniformis juvenile and vermiform adult life stages under in vitro conditions and a second method to collect viable parasitic sedentary females from host plant roots. Rotylenchulus reniformis eggs were hatched over a Baermann funnel and the resultant second-stage juveniles incubated in petriplatescontainingsterilewaterat308C.Nematode developmentwasmonitoredthroughtheappearanceoffourth-stagejuveniles and speci?c time-points at which each developmental stage predominated were determined. Viable parasitic sedentary females were collected from infected roots using a second method that combined blending, sieving, and sucrose ?otation. Rotylenchulus reniformis life stages collected with these methods can be used for nucleic acid or protein extraction or other experimental purposes that rely on life stage–speci?c data.