Hydroponic culture medium was examined for the presence of nematodes that had emigrated from host roots beginning 18 dpi (days post-infection) and at regular intervals thereafter for up to 45 dpi. Unstressed plants yielded only nine male nematodes during this period, corresponding to only 0.18% of the J2 used for inoculum. By contrast, plants that been subjected to pruning stress at 1 or 2 dpi yielded large numbers of motile nematodes (), representing approximately 14% of the inoculum. Examination by stereomicroscopy revealed that the nematodes recovered were all M. incognita
males; no other juvenile or adult stages were seen. These yields of males observed in our study are similar to those previously reported (Davide and Triantaphyllou, 1967
) under nutrient deficiency host stress.
Number of male nematodes recovered from hydroponically grown plants stressed by pruning at various days post infection (dpi). Bars indicate standard error.
Males were found in collections made as late as 45 dpi, although approximately 90% of males were found in the collections made between 18 dpi and 30 dpi. These results confirm previous conclusions that not only does pruning stress induce male sexual development, but also that the response of this stress is limited to the first few days after infection (Triantaphyllou, 1973
). Microscopic analysis revealed that at least 90% of the males generated by this method had both only one gonad and tail spicules, which are diagnostic characteristics of true males and consistent with evidence from previous studies (Papadopoulou and Triantaphyllou, 1982
It has been postulated (Triantaphyllou, 1973
) that the nature of the stress induced by pruning and perceived by Meloidogyne
spp. closely resembles nutrient deficiency. In both conditions, the development of the J2 into an adult is slowed, and the male-to-female ratio is increased due to a switch in the developmental fate of the J2 rather than due to differential death of females. There are many potential physiological and biochemical differences between plants under nutrient deprivation and pruning stress, but the signal for RKN sex determination is apparently common to both conditions. It is still not clear whether this signal is part of a host regulatory pathway (conceivably a single molecule produced by the plant) or a more complex physiological cue perceived by the nematode as a measure of the availability or quality of nutrition, which we term "food signal." Genetic analysis (Lohar and Bird, 2003
) has shown that the ability of RKN to establish successful feeding sites is modulated by the same shoot-derived signaling pathway that regulates the number of nitrogen-fixing nodules induced by rhizobacteria on legumes. However, that study did not reveal altered nematode sex ratios (Lohar and Bird, 2003
). Thus, a more plausible hypothesis of the nature of the cue leading to male development is based on the notion of food signal. In C. elegans,
development of the dauer larva to allow prolonged survival and environmental resistance is triggered by low levels of a general food signal relative to total nematode numbers, which is computed by the C. elegans
LI from the concentration of a constitutively secreted pheromone (Golden and Riddle, 1984
). In fact, this ratio is measured twice during development, demonstrating the functionality of this developmental plasticity throughout development. Meloidogyne incognita
J2 and C. elegans
L2 both express glyoxylate pathway enzymes needed for lipid metabolism characteristic of nematode survival stages (McCarter et al., 2003
). Reentry into the L3 stage from the dauer also is triggered by a general food signal, in this case the reappearance of a relatively adequate food source. Recent evidence demonstrates that C. elegans
L2 also respond to a general food signal for sex determination, where more males develop when grown in log-phase vs. stationary-phase E. coli
(Prahlad et al., 2003
). Understanding the basis for this decision might reveal a mechanism widely used across the phylum.