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24'th Update on Fly Longevity Experiments

Doug Skrecky oberon at vcn.bc.ca
Sun Oct 31 02:56:29 EST 1999


  The 23'rd update of my fly longevity experiments was on August 1998. I
had discontinued these experiments after run #11 was completed, but have
reconsidered and am now back in business.
  For run #11 I tested the effect on fly longevity of various fruit
juices, used in place of the water used with the two controls. The most
outstanding result was the extreme toxicity of Barker's blackcurrent
juice. I have no ready explanation for this, but note that a mixture of
apple and black current juice given to humans had a prooxidant effect on
their plasma proteins. I suspect it was the black current juice which
was the active ingrediant in this mixture. (Am J Clin Nutr 69(1): 87-94
Jan 1999)
  Grape, both red and white grapefruit, and pineapple look modestly
beneficial, but all flies were dead on a day 25 census.

RUN #11         Total # Percent Survival on Day
Supplement     of Flies   6  10  14  21
_______________________________________
cntl 1            (16)   81% 38% 13%  0%
cntl 2            (25)   92  56  40   0
apple             (28)   96  61  18   0
blackcurrent        -     0!  -   -   -
cranberry         (25)   80  52  28   0
grape             (35)   89  71  46   0
grapefruit, red   (42)   90  81  48   7
grapefruit, white (37)   95  81  49   3
orange            (34)   82  65  32   0
pineapple         (26)   81  69  35   4
tomato            (23)   74  39  26   0

  After run #11 all of my breeding bottles for flies independantly
developed a variety of mold infestations that destroyed my breeding
stock. I attributed this unfortunate turn of events to the storage
conditions of the 4-24 fly food, which contains a mold inhibitor. This
may have deteriorated with time, till insufficient levels were still
present to completely inhibit the growth of mold spores. My attempts to
secure an independant supply of mold inhibitor proved to be fruitless,
as I do have the medical degree or the institutional affiliation needed
to obtain these restricted chemicals.
  So I abandoned my attempts to do fly longevity experiments. Time
passed, and eventually I changed my mind, obtained a fresh supply of
4-24 fly food, and fresh breeding stock from the fly lab at a local
university. The results from run #12 were astonishing in the extreme,
with a number of fruit juices proving to double the life span of my
flies. (All fruit juices were from Ceres, with the exception of Knudsen
Black Cherry juice.)  Although the fly food in some of the fruit juice
bottles showed some mild discoloration, a casual examination of the
control bottles did not yield any visual evidence of pathogen growth, so
initially I was rather excited at these results, as follows:

RUN #12        Total #               Percent Survival on Day
Supplement    of Flies   10  26  28  31  35  39  44  48  52  56  60  65
_______________________________________________________________________
cntl 1         (17)      76%  0%  -   -   -   -   -   -   -   -   -   -
cntl 2         (18)      78   0   -   -   -   -   -   -   -   -   -   -
apricot        (10)      70  60  60  60  60  30   0   -   -   -   -   -
black cherry   (15)      80  80  73  73  60  53  47  40  40  20  20   7
guava          (13)      92  69  69  62  62  46  15   0   -   -   -   -
lichi          (12)      67  67  67  67  67  50  33  25  17   0   -   -
mango          ( 8)      63  50  50  50  25  13   0   -   -   -   -   -
papaya         (18)      67  44  44  28  17   6   0   -   -   -   -   -
passion fruit  (16)      81  75  75  75  75  69  56  50  44  31  31   0
peach          (16)      69  63  63  63  50  38  19  13   6   0   -   -
pear           (17)      71  53  47  47  41  24  18   0   -   -   -   -
youngberry     (15)      60  33  33  33  33  20  13  13   7   0   -   -

  The next run issued a warning that all was not well with the fly
experiments. In run #13 all of the bottles were infected with a pink
mold which I believe destroyed the flies long before they had a chance
to grow old and die of old age. This cast a considerable doubt on the
results in run #12. Fruit juices tend to be somewhat acidic, and I
learnt from reference foods on food preservation that acidic pH tends to
inhibit pathogen growth. Moreover 2 months is a typical life span for
the Oregon-R drosophilia flies that I was using. The scientific
literature yields life spans for these from anywhere from 1 month to 3
months at room temperature. Although different sub-strains of Oregon-R
have different life spans the literature notes an impressive cluster of
longevity results at 2 months.
   I finally obtained some sodium benzoate for run #14. Benzoate is a
food preservative that inhibits both bacteria and mold growth,
particularly at acidic pH. I tested 1/32 tsp  of this in 20 gm 4-24 fly
food, in conjunction with various fruit juices  as follows. Keen eyed
readers may spot a minor anomaly in the survival figures.

Run #14            Percent Survival on Day
Supplement       10  20  26  34  40  51  58
___________________________________________
cntl 1          100% 28%  0%  -   -   -   -
cntl 2           92  19   4   0   -   -   -
benzoate (b)     89  83  78  39  18   0   -
apple           100  93  86  36  14   0   -
grape           100  94  81  62  69  31  13
guava           100  88  88  38  13   0   -
guava + b        92  92  92  67  67  17  17
litchi           83  67  58  50  42   0   -
litchi + b       88  88  71  53  29   6   0
mango            87  67  40   0   -   -   -
mango +  b       67  33  25   8   8   0   -
passionfruit     89  84  68  16   0   -   -
passionfruit + b 75  75  25  25  13  13  13

  What I found interesting is the consistancy of the beneficial effect
of benzoate. Maximum survival was always increased, and when combined
with fruit juices the fly food discolourations which occur with time
were eliminated. Only in the control bottles, and the benzoate-only
bottle did the pink slime reoccur, which was associated with a quick
demise of the inhabitants of said bottles. Drosophilia are similar to
vultures in they feed on decaying fruits, so they are naturally
resistant to bacteria, and yeasts. However they are very vulnerable to
molds, and I believe the pink slime I have often observed is such a
mold.
  As negative controls, I included bottles using the same brand of apple
and grape juices as those used in run#11. It is interesting that once
again grape juice improved survival over that obtained with apple juice.
However the huge increase in survival obtained with the fruits juices
used in run#12, were no longer evident, if apple and grape juices are
used instead as the controls. In seems likely therefore that the huge
increases in run #12, were due to an antipathogen effect of the fruit
juices, and not due to any anti-aging effect. Since no visual evidence
for pathogen growth was noted in that run, in the short lived control
bottles, I presume that aged flies are vulnerable to even small amounts
of mold, which are not readily discernible.
  In Run #15 I tested some Libby's (L), Rubicon (R), and Ceres (C) fruit
juices. None of these had benzoate in them, since I had started this run
before the benefits of benzoate had become apparent in run #14. As usual
the control bottles developed a pink slime, and the flies in them died
early. It looked like the Libby's fruit juices had a survival advantage.
Libby also includes citric acid in all their juices, and I wondered
whether this might account for the difference.

Run #15                 Percent Survival on Day
Supplement            16  22  30  36  47  54  71
________________________________________________
cntl 1                79% 67%  0%  -   -   -   -
cntl 2                80  20   0   -   -   -   -
L apricot/mango      100  83  83  58  50  25   0
L peach/passionfruit  71  57  57  57  28   7   0
L pear                94  89  72  56  28  11   0
L strawberry          88  79  58  50  25  17   0
L strawberry/banana   77  77  54  38  23   8   0
R lychee             100  90  40  40  30  20  10
R mango               78  44  67  33   0   -   -
R passionfruit        92  83  50  50   0   -   -
C medley              71  57  29  29   0   -   -
C secrets             75  50  33  17   0   -   -

  Run #16 tested the effect of citric acid in conjunction with benzoate,
as well as higher doses of benzoate. The only bottle that developed pink
slime was one of the 1/32 tsp benzoate bottles. High doses of benzoate
were toxic, but the addition of 1/8 tsp citric acid proved to be safe.

Run #16                Percent Survival on Day
Supplement                    7  24  38  46
___________________________________________
benzoate 1/32 tsp            80%  0%  -   -
benzoate 1/32 tsp           100  81  19  19
benzoate 1/32 + citric acid  91  65  26  13
benzoate 1/16 tsp           100  56  11  11
benzoate 1/8 tsp             92  25   0   -
benzoate 1/4 tsp             71   0   -   -

  Run #17 proved to be a diaster. This was a large run, testing a wide
variety of supplements. I had intended to add citric acid to all of the
bottles, but by mistake I used a bottle of sodium citrate instead, which
does not possess the acidity of citric acid itself. Pink slime destroyed
the flies used in this run, with the exception of a bottle using
passionfruit juice, in place of water, one using a high and toxic dosage
of pyruvate (which is acidic), and bottles with added mustard powder,
which also proved to be toxic, as evidenced by reduced mortality. Homer
Simpson could sum it very nicely. I can only repeat his refrain: Doh!
  Finally Run #18 gets it right. here I tested citric acid, both by
itself, and in combination with benzoate, and passionfruit juice. Here
it is shown that citric acid alone can carry the day, and that neither
benzoate, nor passionfruit juice has any beneficial effects on longevity
in comparison to flies fed a sufficiently high dosage of citric acid.
The only instance in which the fly food showed any discolouration was in
the passionfruit-only bottle, and even here no pink slime was evident.
  In soon-to-be-released follow-up experiments I have used citric acid
as a standard supplement to eliminate pathogens from consideration. In
addition to further longevity experiments, I am also investigating ways
and means for reducing cryoprotectant induced toxicity in flies. I am
hoping that this data may be of some small use in the future development
of usable vitrification solutions for organ cryopreservation.

 Run #18                     Percent Survival on Day
 Supplement                      5   9  12  17  20  25  32  39  43
 citric 1/8 tsp                 79% 63% 58% 37% 26% 11%  5%  0%  -
 citric 1/8 + benzoate 1/32     87  67  60  47  27  20  20   7   0
 citric 1/4                    100  67  74  68  47  26   5   5   5
 citric 1/4 + benzoate 1/32     91  73  73  41  36  23  18   5   5
 passionfruit                   75  50  44  38  13   6   0   -   -
 passionfruit + citric 1/8      76  53  53  35  18  12   6   0   -
 passionfruit + benzoate 1/32   82  71  71  35  24  18  12   6   6
 passionfruit + citric 1/8      55  45  35  25  20  20  15   5   5
              + benzoate 1/32





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