|Abstract:||Nematodes are unsegmented round worms with a pseudocoelomate body cavity. Nematodes are omnipresent in nature. Most are free-living, living as bacterial or fungal feeders, while many nematodes species are parasitic to animals or plants and have a huge impact on human health and agriculture worldwide. Sedentary plant-parasitic nematodes (PPN) like Heterodera glycines, among the most damaging pathogens to agriculture production. The sedentary nematodes are more fecund and considered more damaging than their phylogenetically closest migratory relatives. A better understanding of their pre-hatch and post-infection developmental biology may help to target this economically important pathogen. The embryo development of PPN like H. glycines, less studied in comparison to the free-living nematode Caenorhabditis elegans. H. glycines has complicated hatching behavior. H. glycines hatching increases in the presence of a host plant or hatching stimulants, while some do not hatch in presence of a host. Furthermore, H. glycines females produce approximately one-third of their eggs into an egg sac (egg sac eggs), located at the posterior end of the female, and remaining eggs are retained within the female body (encysted eggs). Different hatching rates have been found between egg sac eggs and encysted eggs. I found that H. glycines develop from a single-cell egg stage to fully formed J2 in approximately seven days. I describe the timeline of H. glycines embryogenesis of encysted eggs and egg sacs eggs in different hatching stimulants. I found that hatching stimulants do not affect embryogenesis timeline. Furthermore, I found that stylet protractor muscles and the primary motor nervous system of H. glycines J2s continue to develop until late in pre-hatch J2s, suggesting their requirement in hatching.
Following infection and the establishment of a feeding site, sedentary nematodes females (H. glycines, Meloidogyne incognita, and Rotylenchulus reniformis) become immobile. Interestingly, loss of mobility after infection is reversed in adult (H. glycines and Meloidogyne incognita) males while females never regain mobility. In C. elegans, contraction and relaxation of most body wall muscles are regulated by motor neurons within the ventral nerve cord (VNC). I studied the VNC neurons of H. glycines, M. incognita, and R. reniformis using DAPI (4′,6-diamidino-2-phenylindole) stain throughout development. In H. glycines, I found a gradual reduction of VNC neurons (65 to 40) during development from the mobile J2 to sedentary J3 and J4 females. Some nuclei of the VNC in sedentary stages were located several microns away from the ventral midline. Strikingly, I found 70 neurons in the adult male VNC and a reorganization of the cord into a linear arrangement. Similar to H. glycines I found fewer VNC neurons in sedentary stages of M. incognita and R. reniformis in comparison to mobile stage. The number of VNC neurons remained stable in P. penetrans, a migratory nematode. My results suggest that VNC motor neuron degeneration is correlated with sedentary behavior.
Unlike most nematodes, sedentary nematode females grow disproportionately greater in width than in length developing into a saccate shaped adult. In C. elegans, body size is correlated with stem-cell-like divisions of laterally positioned stem cell-like ‘seam’ cells that contribute to an increase in the total number of epidermal nuclei. I examined the epidermis of both live and fixed H. glycines at regular time points after synchronized infection. First, I confirmed the presence of seam cells in H. glycines. Then I found that in post-infection H. glycines seam cells proliferate extensively during each developmental stages. I found that H. glycines adult female epidermis comprises a syncytium of approximately 1800 epidermal nuclei, comparison vermiform species C. elegans approximately 140. Saccate shaped development has evolved multiple times among nematodes. To study the evolution of the saccate shape nematode, I study two other saccate shape nematodes, M. incognita and R. reniformis. H. glycines and M. incognita are phylogenetically distinct but have similar life cycles, whereas H. glycines and R. reniformis are phylogenetically close but have a different life cycle. I found that M. incognita seam cell also proliferates following infection, however, the pattern of the division was different than in H. glycines. Both H. glycines and M. incognita epidermal nuclei were polyploid. Interestingly, R. reniformis does not show increased seam cell proliferation compared with C. elegans.
My results on H. glycines hatching stimulants provide insight into the primary survival mechanism for this important parasite. I have found that VNC neuron degenerates in sedentary plant-parasitic nematode species. The nervous system of nematodes has been the primary target for nematode control. A better understanding of how the nervous system regulates the specific behavior may be used to develop targeted control strategies for these economically important plant-parasitic nematodes while avoiding off-target effects to beneficial nematodes and humans. My results on sedentary nematodes epidermis suggest distinct mechanisms evolved to produce a similar phenotype from a common ancestor. More information on sedentary plant-parasitic nematode epidermis may provide better insight into the evolution of these sedentary nematodes.