Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2013
Publication Date: 6/1/2013
Citation: Ganji, S., Wubben, M., Jenkins, J.N. 2013. Two simple methods for the collection of individual life stages of reniform nematode, Rotylenchulus reniformis. Journal of Nematology. 45:87-91.
Interpretive Summary: The reniform nematode is a serious pest of cotton and soybean in the southeastern United States. The life-cycle of the reniform nematode is very different from that of other sedentary plant-parasitic nematodes. For example, cyst and root-knot nematode juveniles immediately begin to infect plant roots after hatching from eggs in the soil and complete their development into adult life-stages while feeding in the root. In contrast, reniform nematode juveniles do not infect after hatching but instead become immobile in the soil until their development into vermiform, i.e., worm-like, males and females is complete. In order to perform a comprehensive assessment of a specific nematode gene, it is necessary to determine the gene’s expression in all life-stages of the nematode. Protocols designed to isolate life-stages of cyst and root-knot nematodes cannot be used to isolate the juvenile life-stages of reniform nematode. This paper presents a method that can be used to isolate large numbers of reniform nematode from each life-stage so that the developmental expression of a gene can be determined. The ability to identify in which life-stage(s) a gene is expressed will help us determine which genes may be suitable targets for biotechnology-based control methods. This protocol may also be used to isolate life-stages for testing potential new chemical control measures.
Technical Abstract: 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 plant hosts has increased; however, the unusual lifecycle of R. reniformis presents a unique set of challenges to researchers who wish to study the developmental expression of a particular nematode gene or evaluate life stage-specific effects of a specific treatment such as RNA-interference or a potential nematicide. Unlike other sedentary plant-parasitic nematodes, the R. reniformis juvenile stages do not feed; only the vermiform adult female infects the host root. In this report, we describe a simple method that allows the collection of R. reniformis juvenile and vermiform adult life stages under in vitro conditions. R. reniformis eggs were hatched over a Baermann funnel and the resultant second-stage juveniles incubated in petri-plates at 30°C. R. reniformis juvenile development was monitored over time and the presence of residual intact juvenile cuticles was used to identify the third-stage and fourth-stage juveniles at specific time-points. Parasitic sedentary females were collected from roots of infected host plants by a combination of blending, sieving, and sucrose floatation. The methods presented here provide a way to collect large quantities of R.reniformis life stages that can be used for nucleic acid or protein extraction or for other experimental purposes.