Investigation of the regulation of transcriptional changes in Ancylostoma caninum larvae following serum activation, with a focus on the insulin-like signalling pathway.
The exit from dauer in the free-living nematode Caenorhabditis elegans is under the control of a single amphidial neuron (ASJ) of the insulin-like signalling pathway. Mutations of this pathway have the ability to suppress entry into the dauer stage. It has been postulated that insulin-like signalling plays a significant role in the response to serum stimulation in vitro of the third-stage larvae (L3s) of the canine hookworm Ancylostoma caninum. To test for the possible involvement of the insulin-like signalling cascade in the response to serum stimulation, the effects of two signalling stimulants (8-bromo cGMP and arecoline) and four inhibitors, namely 4,7-phenanthroline, phosphoinositide-3 kinase (PI3K), Akt inhibitor IV and rapamycin on feeding and on levels of selected activation-associated mRNAs in serum-stimulated L3s were explored. L3s of A. caninum were pre-incubated with or without the appropriate inhibitor/agonist. Following serum-stimulation, the feeding activity was assessed. The transcription levels of a number of activation-associated mRNAs linked to particular expressed sequence tags (ESTs) were investigated by reverse transcription, real-time PCR (rtPCR). The treatment of worms with 4,7-phenanthroline completely suppressed feeding and significantly reduced the differential levels of most activation-associated mRNAs, whereas the treatment with cGMP resulted in the resumption of feeding in almost 85% of the L3s and yielded a specific transcriptional profile consistent with that following serum stimulation. The treatment of L3s with arecoline resulted in the resumption of feeding in approximately 85% of L3s, but did not result in a transcriptomic profile consistent with activation. A complete reduction in feeding was recorded in the presence of the PI3K inhibitor LY294002 (1mM) and resulted in a pronounced dampening of differential transcription in response to serum stimulation for the molecules examined. Akt inhibitor IV resulted in a approximately 70% reduction in feeding but had almost no effect on the level of any of the activation-associated mRNAs studied. Rapamycin was shown to have a weak effect on feeding, and several of the mRNAs studied exhibited greater than expected transcription following treatment. The complexities of activation-associated transcription could not be addressed using the current approach. A larger number of mRNAs needs to be investigated in order to predict or identify regulatory mechanisms proposed to function in the insulin-like signalling pathway in A. caninum.
Datu
Bennett J D
BJ
Helminth Biology Laboratory, Division of Infectious Diseases, Queensland Institute of Medical Research, 300 Herston Road, Herston, Queensland 4006, Australia.
Loukas
Alex
A
Cantacessi
Cinzia
C
O'Donoghue
Peter
P
Gasser
Robin B
RB
D000722:Ancylostoma / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D001115:Arecoline / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D051058:Oncogene Protein v-akt / Q000037:antagonists & inhibitors; D010618:Phenanthrolines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D044967:Serum; D015398:Signal Transduction / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology; D014158:Transcription, Genetic / Q000502:physiology*
D015801:Helminth Proteins; D007328:Insulin; D010618:Phenanthrolines; C053487:4,7-phenanthroline; D001115:Arecoline; D019869:Phosphatidylinositol 3-Kinases; D051058:Oncogene Protein v-akt; D006152:Cyclic GMP; D020123:Sirolimus