W. F. LOOMIS 349 



The next step was to expose Hydra to BVC solutions whose 

 PCO2 had been raised artificially. Table 1 records the result of this 

 1957 experiment, an experiment that has been found to be highly 

 repeatable as described above. Taking both the group and stag- 

 nation factors of this particular experiment into account, it is clear 

 that pCOo strongly affects the reaction. Just as clear, however, is 

 the fact that an increase in pCO^ is not sufficient, for Hydra main- 

 tained in a flowing stream of BVC (Fig. 4) whose pCO^ varied 

 from 0.03% to 10% failed to differentiate sexually. Similar exposure 

 of Hydra to conditions of both high pCO^. and low pO^ failed to 

 induce sexual differentiation, and it was concluded that a third 

 factor must be operative in the system ( 19 ) . 



3) pNH, 



Evidence that a third factor existed induced us to assay samples 

 of "crowded water" for such metabolic gases as carbon monoxide, 

 methane, ethylene, H^S, SOo etc. Analysis by infra-red spectography, 

 mass spectography and gas-liquid partition chromatography failed 

 to show such gases to be present, only CO^. and NH,-, being detect- 

 able beyond the gases found in normal air. Analysis for NH3 by 

 the 1 ml. syringe method showed that sexual cultures usually con- 

 tained about 1 mg.T. NH... and that Hydra secreted large amounts 

 of ammonia after being fed with brine shrimp. Since the toxic level 

 of NH3 varied with the pH, it was concluded that the active species 

 was the NH3 molecule rather than the NH4+ ion as only the fonner 

 could penetrate the lipid cell membrane which is largely imper- 

 meable to polar solutes such as NH4+ (27). 



Exposure of Hydra to increased levels of pNHo was accom- 

 plished by adding different amounts of NH4OH to buffered culture 

 solutions, and it was found that this variable alone and in various 

 combinations with increased pC02 and decreased pO^ was unable 

 to induce sexuality in Hydra at least under the conditions tried to 

 date. One insight came from these experiments, however: it became 

 clear that Hydra release the salt ammonium bicarbonate into their 

 halo zone and that this buffer is equivalent to NaHCOg which, 

 as we will see below, strongly affects the system. 



