Axiocam compound microscope

Pasteuria endospores were produced on Adzuki bean with single egg-mass culture of M. incognita as described by Rao et al. (2012) (link). To test the effect of dsRNA feeding on attachment of P. penetrans (Strain AII-329: Pasteuria collection, ICAR-IARI, New Delhi, India) endospores to the J2 cuticle surface, 200 dsRNA-treated J2s were removed from the dsRNA soaking solution and mixed with 100 μl suspension of spores (2.5 × 10³ ml^-1) and centrifuged at 6000 G for 3 min following the method of Hewlett and Dickson (1993) (link). The endospore adhesion was quantified by removing 30 J2s from the suspension after 30 min and spore attachment observed using a Zeiss Axiocam compound microscope and photographed. All assays were performed in triplicate and freshly hatched juveniles were used as control.

Gene-specific primers (miFARp_F and miFARp_R) were used for localization of expression of Mi-far-1 mRNA by performing in situ hybridization as described in Kimber et al. (2002) (link). Around 20,000 J2s were concentrated to a 10–15 μl pellet and fixed in 2% paraformaldehyde at 4°C for 18 h, followed by 4 h incubation at room temperature and cut into sections. The gene specific primers miFARp_F and miFARp_R were used to amplify DIG-labeled sense and antisense RNA probes (Roche, Germany) from cDNA of M. incognita J2s. DIG-labeled sense or antisense RNA probe was added to the hybridization solution containing the nematode sections, and then rotated at 50°C for 12 h. After hybridization, the nematodes were stained and photographs were taken with Zeiss Axiocam compound microscope.