100 CARNEGIE INSTITUTION OF WASHINGTON. 



each control and test bottle contains equal numbers of young from the same 

 brood and handled precisely alike, except the specific treatment given the test. 



"Some of the main points of attack and accomplishment upon this problem 

 may best be set forth by the use of a table showing some of the data. (These 

 tabulated data are subject to subsequent verification.) The treatments may 

 be divided into four groups: (1) aeration, to change the content of volatile 

 substances in the culture- water or in the animals themselves; (2) change in 

 the food directly; (3) treatment )^ith excretory products or related substances; 

 and (4) treatment with other substances. The aeration experiments were 

 conducted on the Supposition that the accumulation of excretory products and 

 possibly a reduction of oxygen supply in the crowded bottles might be factors 

 in bringing about the production of males. The aeration of the animals them- 

 selves was accomplished by exposing them to the air for a time during their 

 'critical period' in a thin film of water on a microscopic shde or in a Syracuse 

 dish. Such aeration reduced but did not eliminate the production of males, 

 inasmuch as one-third as many males were produced by the aerated mothers 

 as by those not so treated. In other series of experiments, the test animals 

 were reared in the same quantity of culture-water as the controls, but in a 

 wide covered dish, so that the culture-water was only 3 mm. in depth, thus 

 providing much greater aeration than in the control bottles. Such treated 

 mothers produced about half as many males as the control mothers. In 

 other large series of experiments air and, in still others, oxygen was bubbled 

 through the test bottles. Approximately three-fifths as many males were 

 produced in such treated bottles as in the controls. On the other hand, bub- 

 bling with nitrogen, in which case the bubbling was much less extensive, 

 reduced the male production, but to a less extent than bubbUng with 

 air or oxygen. This suggests that scarcity of oxygen is not a faetor, since 

 the bubbling of nitrogen materially reduces the oxygen content of the treated 

 bottles, and further suggests that, since the bubbling was less extensive and 

 consequently removed less carbon dioxide or other volatile excretory products, 

 volatile excretory products are factors in male production. 



"Scarcity of food as a possible factor in male production is eliminated 

 by experiments in which crowded mothers in dilute food are shown to pro- 

 duce only two-fifths as many males as those in the regular food solution, 

 notwithstanding the fact that average size of the broods of young was only 

 9 for the dilute food and nearly 20 for the control. However, variation in 

 the culture medium is shown to be a factor in male production, since animals 

 reared in water containing algae as food for the animals (instead of bacteria as 

 in our manure culture solution) produce fewer males than those reared in 

 the controls and fed upon bacteria (in the manure solution). Further, the 

 age of the culture medium or of the manure used in making it up is a factor in 

 male production, as shown by the data in line 8 of the table, the newer material 

 producing many times more males than old material. These last experiments 

 are of interest as accounting for the varied male percentages shown in the 

 controls of the different series tabulated. 



"Treatment with various amounts of excretory products, including am- 

 monium hydrate, urea, urine, and chicken manure, reduced the percentage 

 of males, though treatment with uric acid and carbon dioxide showed a some- 

 what greater percentage of males in the test bottles. The other agencies 

 tabulated here (pressure excepted) reduced the percentage of males. The 

 few experiments conducted in which the animals were subjected to pressure 

 produced many more males in the tests than in the controls. Further tests 

 of this are necessary, but it would seem that pressure treatment may be added 

 to uric acid and carbon dioxide treatment as agencies increasing male pro- 

 duction. 



