Chapter 19
Lab work suggests that pre-zygotic and post-zygotic fertility regulation are both mediated by methylation, raising the question of whether current folic acid use is safe.

Previous work with fruit flies had suggested that kinship does indeed change fertility in these insects and that the change is so rapid and cyclic that it cannot be due to DNA mutations but must be brought about by an epigenetic mechanism.  Let me unburden myself of everything I know about epigenetics.  1) Epigenetic markers are important in embryonic and subsequent growth and development.  2) Methyl groups (A methyl group is a carbon with three hydrogens attached and an open bond with which it can combine with other things.) have specific sites on DNA, where they can bond, usually reducing the function of an enzyme. 3)  Ethyl groups can also bond to DNA.  4) There are other epigenetic processes, but I have no clue what they are or how they work. 
We shall exploit the fact that methyl groups on DNA can alter the function of genes.  Accordingly, I made a cocktail that was reported by Waterson 31.   
I took 30 grams choline, 30 grams betaine, 30 milligrams folic acid, 3 milligrams vitamin B12, 15 grams l-methionine and 30 grams of zinc made up to 1 liter with water.  The pills did not dissolve completely, so there was a lot of mixing to assure a consistent content.  I called the full strength 100% and did dilutions from there.  I found in a side experiment that the flies could tolerate a 40% dilution, but not much more.  Waterson apparently worked up to a level where the methyl receptors were saturated.  In flies this might have produced a maximum fertility, but that would have been hard to interpret.  So, I did a control baseline, during which the flies underwent a complete cycle of damped oscillation and then gave them a 40% dilution, reducing the population over time.  Daily counts were made in two windows of the cage and then added together every 2 weeks.

The schedule was table 1, figure 56:
Table 1

Daily counts in two windows continued and the results pooled over 2-week intervals were examined.  Concentrations and dates they were given are shown.
Fig. 58
The result is shown in figure 59.

Graph of the time course of a population of fruit flies under condition of decreasing concentration of a methylating cocktail.  The vertical axis is biweekly counts in the windows of the cage.  The horizontal axis is the concentrations as shown in the table of figure 58.  Highest and lowest counts shown.
Fig. 59
The first observation is that yes, methylation seems to have an effect on fertility.  My first impulse was to give 90% confidence intervals, but since there is no certainty that the data are distributed on a normal curve, it seemed clearer at every two-week interval to give an average, a highest value and a lowest value. 
There are a number of things that might be going on: 1) there might be a toxic effect from the supplements.  2) There might be a nutritional effect from the supplements.  3) The dilutions are changing.  4) There is definitely an underlying post-zygotic process.  However, even using all three I do not see how this complex response could be caused. 
Until someone comes up with a better explanation, we are left with the weight of the evidence indicating that there is at least one pre-zygotic mechanism and at least one post-zygotic mechanism and both of these are mediated by a process involving methylation.
Having found the weight of evidence to suggest that methylation drives fertility in fruit flies, we must conclude that the statement, “Only kinship determines fertility, minus rare disaster,” is incomplete.  One must add, “And methylation has an effect.” 
Pursuing the issue further, I wondered if the entire cocktail used in the previous experiment was actually needed.  Would folic acid alone have an effect?  In the earlier study, the cocktail seemed to have a significant effect over an entire order of magnitude of dilutions.  Doing the same study with only folic acid would have taken years.
But in fact, there is one concentration of folic acid that is of the greatest importance.  Women who are pregnant or liable to become pregnant are advised to take folic acid regularly.  Received wisdom is that since folic acid is water soluble; it does not accumulate in the body so large doses are given with impunity.  Now folic acid is not itself a natural food, but is thought to substitute for “folate” compounds, three micrograms of folic acid being the equivalent of five micrograms of folate.  That dose of folate is about what is found in an apple, if eaten seeds and all.  So, three micrograms of folic acid, or one ripe apple daily, should be a pretty good amount even if the rest of the diet is lacking.
“Should be” is not “is,” and what I am about to say must not be taken as advice but as an inducement to further study.
Folate is a necessary vitamin.  Its lack can lead to a lethal anemia, since it is necessary for DNA replication, which is necessary for cell division.  Moreover, a lack of folic acid is correlated with a condition called a neural tube defect in a developing pregnancy.  The condition can range in severity from an almost trivial sinus tract in the small of the back to a devastating neurological malformation.  The condition can be prevented by folate about half the time. 
So current practice is to give a woman hundreds of micrograms of folic acid a day.  Please do not eat a hundred apples a day seeds and all; that would include enough cyanide to kill you even though cyanide is nicely soluble in water. 
The pressing question is whether the current recommended dose of folic acid is safe.
Previously I had no idea what dose of the cocktail had an effect, and it took years to look at a range of values.  But now I could focus on a single dose of a single chemical.
As best as I could, I prepared two feeding formulae for the flies.  One was the standard recommended by the supplier.  The other was mixed not in straight water, but in water that had been given folic acid.  The amount was chosen such that the final food would have the same amount of folic acid per calorie as a woman eating a good 1,800 calorie diet, supplements and all, would get. 

I’ll not give you the actual number of folic acid pills I used.  Figure it out for yourself if you choose to run the experiment, which I most urgently beg you to.  The routine was to add four males and four females to a vial and follow them for two weeks.  After about 11- or 12-days fresh flies would begin to emerge.  As soon as they did, I would daily shake them out into a vial to be kept giving them normal food.  There were three control vials and three fortified. 
My method of transfer was primitive but consistently applied.  I would take an old vial and a fresh one and a new stopper, the old vial already being stoppered.  I would hold both vials in my left and tap them on the desk top while I removed the old stopper.  I would take the new vial in my right hand, continuing to tap both and then would quickly invert the new vial, placing it top to top on top of the old vial.  Keeping the two together I would invert them both.  Then, still tapping, I would put the old vial back in my left and, replace the old stopper and place the new one.  I had maintained the line of wild type flies for many years.  There were two obvious limitations.  Not all the flies got transferred to the new vial; if I got stubborn and tapped very hard during transfer, the old food with maggots and flies would drop into the new vial, making a mess and necessitating discarding the line.  The other limitation is that fly husbandry calls for starting each line with four males and four females or with twenty random flies; my technique transferred 50, to 100 which was too many for optimal fertility, but with sixteen lines I was carrying, I was looking for the quick way out.
At the end of two weeks, all the flies that had emerged from all the control vials were added together and all that had emerged from the treated vials likewise and are reported below. 
I ran the flies, all six vials, in the control medium for a couple generations with the standard four male, four female start each generation and then started adding the spiked water to half of them.  What I got is shown in figure 60.

Population trajectory of fruit flies.  The horizontal axis is five successive generations of flies.  The vertical axis is the sum of daily counts as described in the text.  Treated flies line B.  Control line A.
Fig. 60
It seems to me that the folic acid reduces fertility.  In terms of the computer program it is increasing the rate at which sites that affect pre- and/or post-zygotic fertility change with a resulting increase in the difference between critical areas on chromosomes and thus reducing the number of offspring.  The fertility increases both in the control and treated lines as the improved husbandry increases fertility overall. 
It would be highly desirable to do the same study in mice.  Fruit flies often eat fruit and might therefore have developed at least as good a defense against excess folate as humans.  On the other hand, mammals are more complex and might have developed a better resistance to folate overdose.  There is no way I can think of that would let us know without doing the experiment. 
It's all very grim, so let me end on an (an eminently ignorable) lighter note.  My brother was touring Israel.  About sundown, the tour guide took them to a ridge overlooking a valley called Mitzpe Shalem after a geologist; in fact, Shalem means twilight.  The guide explained that this valley had been sacred to the ancient Canaanite god El, god of twilight and peace.  As they all stood there, my brother heard a low-pitched sound, somewhere between speech, song and a rumble.  He called the guide over, but by the time the guide got to him the sound had ceased.  My brother asked what the sound was.  The tour guide became most upset, “freaked out,” declared that there was no sound and insisted the others in the group not be told.  It was just another impossible thing.  Maybe it was a breath of air moving over a landscape feature in the distance.  But these things do happen.  No matter how terrible it all looks, there just might be another impossibility coming out of nowhere so that we manage to survive and become better than we are. 

Does El still call?
Figure 61 taken from Google Earth Pro.  The original is in color.  I have sought but not received permission to alter the image. 

Chapter 20

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