Phosphoinositide-3-OH-kinase inhibitor LY294002 prevents activation of Ancylostoma caninum and Ancylostoma ceylanicum third-stage infective larvae

The developmentally arrested hookworm infective larva resumes development only after encountering specific host-mediated cues during invasion. These cues activate a signaling pathway that culminates in the resumption of development. In Ancylostoma caninum, activation is characterised by the resumption of feeding and the release of excretory/secretory products required for infection. The dauer stage of the free-living nematode Caenorhabditis elegans is a developmentally arrested stage analogous to the hookworm infective larva. Dauer larvae exit developmental arrest in response to environmental cues that indicate favorable conditions for reproduction and growth. Because of the similarity between dauer recovery and activation, exit from dauer provides a model for hookworm larval activation. An insulin-signaling pathway has been implicated in controlling exit from developmental arrest in both C. elegans dauers and A. caninum larvae. To further investigate the role of insulin signaling in hookworm larval activation, the phosphatidylinositol-3-OH kinase inhibitor LY294002 was tested for its effect on in vitro activation using the resumption of feeding as a marker for activation. LY294002 prevented feeding in A. caninum infective larvae stimulated with host serum filtrate and a glutathione-analogue, the muscarinic agonist arecoline, or the cell permeable cGMP-analogue 8-bromo-cGMP. Similar results were seen with the congeneric hookworm Ancylostoma ceylanicum. These data suggest that an insulin-signaling pathway mediates activation in hookworm larvae, as in C. elegans, and that the phosphatidylinositol-3-OH kinase inhibitor acts downstream of the cGMP and muscarinic signaling steps in the pathway. In A. caninum, LY294002 had no effect on the release of excretory/secretory products associated with activation, suggesting that the secretory pathway diverges from the activation pathway upstream of the phosphatidylinositol-3-OH kinase step. These results provide additional support for the insulin-signaling pathway as the primarily pathway for activation to parasitism in hookworm larvae.     

Effects of age and liquid holding on the UV-radiation sensitivities of wild-type and mutant Caenorhabditis elegans dauer larvae

The dauer larva is a facultative developmental stage in the life cycle of the nematode Caenorhabditis elegans. Dauer larvae, which can survive under starvation for over 60 days, resume normal development when feeding is resumed. Wild-type (N2) and 4 radiation-sensitive (rad) mutant dauer larvae were tested for their abilities to develop into adults after UV-irradiation. The rad-3 mutant was over 30 times as sensitive as N2; rad-1, rad-2 and rad-7 mutants were not hypersensitive. Irradiation also delayed development in survivors. Wild-type dauer larvae did not differ in radiation sensitivity from 0 through 50 days of age. There was no liquid holding recovery (LHR); that is, survival did not increase when wild-type dauer larvae were held in buffer after irradiation.     

The second messenger cyclic GMP mediates activation in Ancylostoma caninum infective larvae

The developmentally arrested infective larva (L(3)) of hookworms encounters a host-specific signal during infection that initiates previously suspended developmental pathways. Activated L(3) express a parasitic gene set that encodes proteins involved in moulting, growth and development to the adult stage. Early events in this activation to parasitism can be investigated using an in vitro larval feeding assay. When Ancylostoma caninum L(3) are exposed to a host-like stimulus, they resume feeding and release molecules involved in infection. The dauer larva of the free-living nematode Caenorhabditis elegans is a developmentally arrested stage analogous to the hookworm L(3). Recovery from the dauer stage has been proposed as a model for the transition to parasitism in hookworm. Dauer formation and recovery involve several tightly regulated pathways, including a cyclic GMP mediated signalling pathway. To determine if hookworm L(3) activation uses a similar pathway, 8-bromo-cyclic GMP, a membrane permeant analogue of cyclic GMP, was tested for its ability to stimulate feeding. Populations of L(3) incubated with 0.5 mM 8-bromo-cyclic GMP began feeding, and reached maximum feeding at 3.5-5.0 mM. Unlike the serum stimulus, which triggers feeding after a short exposure, 8-bromo-cyclic GMP must be present throughout the entire incubation. Both serum stimulated and 8-bromo-cyclic GMP stimulated L(3) secreted Ancylostoma secreted protein 1, indicating that the stimuli activate the same pathway. Serum and 8-bromo-cyclic GMP stimulated feeding was inhibited by atropine, a muscarinic receptor antagonist. However, only serum stimulated feeding was inhibited by 4,7-phenanthroline, a non-chelating isomer of the metalloprotease inhibitor 1,10-phenantholine. The results indicate that cyclic GMP mediates activation in hookworm larvae, and that a muscarinic receptor is involved in activation. This also suggests that hookworm activation and dauer recovery share similar signalling pathways, and that C. elegans dauer recovery can be used as a model for the transition to parasitism in hookworms.     

Mutants of Caenorhabditis elegans that form dauer-like larvae

The development, ultrastructure, and genetics of two mutants that form dauer-like larvae have been characterized. Dauer larva morphogenesis is initiated regardless of environmental stimuli, and it is incomplete or abnormal. The resistance to detergent characteristic of normal dauer larvae is not fully achieved, and the mutants are unable to exit from the dauer-like state of developmental arrest. Mutant life span is not extended beyond the three weeks characteristic of the nondauer life cycle, whereas normal dauer larvae can live for several months. Growth of daf-15(m81)IV, the less dauer-like of the two, is nearly arrested at the second (dauer-specific) molt, but feeding is not completely suppressed. Head shape, cuticle, and intestinal ultrastructure are nondauer, whereas sensory structures (amphid and deirid) and excretory gland morphology are intermediate between that of dauer and nondauer stages. The daf-9(e1406)X mutant is dauer-like in head shape, cuticle, and deirid ultrastructure, intermediate in amphid and inner labial neuron morphology, and nondauer or abnormal in the intestine. Also, the daf-9 mutant exhibits abnormalities in the pharyngeal arcade cell processes and pharyngeal g1 gland. Double mutants carrying both daf-9 and daf-15 are more resistant to detergent than either single mutant. Like the single mutants, they cannot complete morphogenesis, and they are unable to exit from the dauer-like stage. Both daf-9 and daf-15 mutations are epistatic to previously described dauer-defective mutations, indicating that these two genes act late in the pathway leading to the dauer larva. The genetic tests and the mutant ultrastructure suggest that the two genes may affect parallel pathways of morphogenesis.     

Exposure to the metabolic inhibitor sodium azide induces stress protein expression and thermotolerance in the nematode Caenorhabditis elegans

Historically, sodium azide has been used to anesthetize the nematode Caenorhabditis elegans; however, the mechanism by which it survives this exposure is not understood. In this study, we report that exposure of wild-type C elegans to 10 mM sodium azide for up to 90 minutes confers thermotolerance (defined as significantly increased survival probability [SP] at 37 degrees C) on the animal. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed enhanced Hsp70 expression, whereas Western blot analysis revealed the induction of Hsp16. We also tested the only known C elegans Hsp mutant def-21 (codes for Hsp90), which constitutively enters the stress-resistant state known as the dauer larvae. Daf-21 mutants also acquire sodium azide-induced thermotolerance, whereas 3 non-Hsp, constitutive dauer-forming mutants exhibited a variable response to azide exposure. We conclude that the ability of C elegans to survive exposure to azide is associated with the induction of at least 2 stress proteins.     

Fine structure of the Caenorhabditis elegans secretory-excretory system

The secretory-excretory system of C. elegans, reconstructed from serial-section electron micrographs of larvae, is composed of four cells, the nuclei of which are located on the ventral side of the pharynx and adjacent intestine. (1) The pore cell encloses the terminal one-third of the excretory duct which leads to an excretory pore at the ventral midline. (2) The duct cell surrounds the excretory duct with a lamellar membrane from the origin of the duct at the excretory sinus to the pore cell boundary. (3) A large H-shaped excretory cell extends bilateral canals anteriorly and posteriorly nearly the entire length of the worm. The excretory sinus within the cell body joins the lumena of the canals with the origin of the duct. (4) A binucleate, A-shaped gland cell extends bilateral processes anteriorly from cell bodies located just behind the pharynx. These processes are fused at the anterior tip of the cell, where the cell enters the circumpharyngeal nerve ring. The processes are also joined at the anterior edge of the excretory cell body, where the excretory cell and gland are joined to the duct cell at the origin of the duct. Secretory granules may be concentrated in the gland near this secretory-excretory junction. Although the gland cells of all growing developmental stages stain positively with paraldehyde-fuchsin, the gland of the dauer larva stage (a developmentally arrested third-stage larva) does not stain, nor do glands of starved worms of other stages. Dauer larvae uniquely lack secretory granules, and the gland cytoplasm is displaced by a labyrinth of large, transparent spaces. Exit from the dauer stage results in the return of active secretory morphology in fourth-stage larvae.     

Developmental regulation of energy metabolism in Caenorhabditis elegans

Changes in energy metabolism during larval development in Caenorhabditis elegans have been investigated using phosphorus nuclear magnetic resonance (31P NMR). The relative concentrations of ATP, ADP, AMP, sugar phosphates, and other metabolites were observed to change during larval development, producing stage-specific spectra. These spectra are consistent with enzyme assays for isocitrate dehydrogenase and isocitrate lyase, indicating that high activity of the glyoxylate pathway during embryonic development decreases during the first larval (L1) stage, and respiration during the L2, L3, and L4 stages occurs preferentially through the TCA cycle. Metabolic strategies were further studied using mutants that are predisposed to enter the dauer stage, a developmentally arrested third-stage larva formed under conditions of overcrowding and limited food. After the L1 molt, energy metabolism in animals destined to become dauer larvae diverges from that of animals committed to growth. Relative to the L1, the L2 larvae committed to growth exhibit increased isocitrate dehydrogenase activity as well as increases in ATP and other high-energy phosphates, but predauer (L2d) larvae exhibit declining enzyme activities and declining levels of high-energy phosphates. The predominant phosphorus NMR signal in dauer larva extracts corresponds to inorganic phosphate. We conclude that metabolism is regulated during C. elegans larval development, with a major transition apparent after the L1 stage. This transition does not occur in larvae destined to form dauer larvae.     

Liquid culture of the entomopathogenic nematode-bacterium-complex Heterorhabditis megidis/Photorhabdus luminescens

A process technology for production of the entomopathogenic biocontrol nematode-bacterium complex Heterorhabditis megidis/Photorhabdus luminescens in monoxenic liquid culture in laboratory scale bioreactors is described. Dauer juvenile yields varied between 21 and 68 million dauer juveniles/l medium. The maximum density was reached at 13 to 25 days after inoculation of P. luminescens. The reason for the high variability in yield was identified. After the 24-h bacterial preculture the bioreactor is inoculated with nematode dauer juveniles which develop to self-fertilizing hermaphrodites. The exit from enduring dauer stages (recovery) was between 18 and 90% of the inoculum density. Low dauer juvenile recovery resulted in the development of two-generations within 20 to 25 days. In contrast, high dauer juvenile recovery led to a one-generation process terminated within 15 days. Factors influencing dauer recovery are still unknown.