STATE PKANT BOARD
E-488 September 1939
United States Department of Agriculture
Bureau of Entomology and Plant Quarantine
THE LABORATORY-FIELD METHOD FOR TESTING CODLING MOTH INSECTICIDES
By L. F. Steiner, Division of Fruit Insect Investigations
The laboratory-field method for testing codling moth insecticides,
described herein, consists essentially of the subjection of field-sprayed
fruit to the attack of a known population of codling moth larvae under con-
ditions that are controlled after attack begins. Its primary objective is
to determine the relative larvicidal efficiency of various spray treatments
at frequent intervals during the season, and to evaluate separately the
influence of the many factors other than codling moth abundance that deter-
mine the control value of an insecticidal treatment. It is designed to fill
in the gap between the initial testing of a spray treatment under purely
laboratory conditions and its final testing and demonstration in the field
on a large-scale, commercial basis.
The general idea of this method was proposed by E. H. Siegler in 1929
for use with the so-called apple-plug method, in a mimeographed statement
circulated among certain interested workers, and was discussed by Siegler
and Munger in a paper published in 1933 (5).2 The method was further
developed by Lathrop and Sazama (4) in 1931 and since then has been in use
each year at the Vincennes, Ind., laboratory of this division. Subsequent
\ to 1935 the major part of the work with insecticides there has been carried
on in this manner.
The establishment at Vincennes in 1934 of a chemical laboratory of the
Division of Insecticide Investigations for cooperative work has made it
possible to obtain a type of information which ties in to better advantage
with the laboratory-field method than with any other known method of testing
codling moth sprays. The Division of Fruit Insect Investigations records in
detail meteorological conditions at the time of spraying and daily between
spray applications, determines the larvicidal efficiency and other bio-
logical effects of the sprays, and makes records of spray injury and the
damage caused by other insects and diseases. The Division of Insecticide
Investigations measures the quantity of spray deposits present, determines
the growth rate of the fruit in the experiments, records water hardness and
occasionally hydrogen-ion concentration, and also makes analyses of the
1 This is a summary of a paper presented on December 2, 19%8, at a confer-
ence of Division leaders and others of this Bureau, held at Washington, D. C.
a Numbers in parentheses refer to Literature Cited, p. 10
Basis for Selection of Treatments
Treatments are selected on the basis of leads developed during the
laboratory-field tests of the previous season. The selection is also guided
by the results of extensive laboratory spray tests, usually conducted in the
winter in cooperation with the Division of Insecticide Investigations. From
50,000 to 100,000 larvae are used annually in such preliminary tests. Most
of the work has been with fixed nicotines, phenothiazine, lead arsenate,
calcium arsenate, and occasionally with new materials.
Plot Lay-out and Spraying Methods
Plots are laid out regardless of moth-population differences in the
orchard. Care is taken to avoid orchards that are closely planted and in
which, consequently, there would be danger of spray drift. As level a block
as possible is obtained, in order that undue interplot variations in tem-
perature, relative humidity, and wind velocity may be avoided. The set-up
is so arranged that all plots can be sprayed the same day unless rains or
For the main experiments Grimes and Jonathan trees 19 to 23 years old
have been employed. Enough trees (usually 4 to 8) are employed to provide
plenty of fruit for sampling. This may run up to 3,000 apples on certain
plots where both biological and chemical data are to be obtained.
Sprays are applied with equipment and at pressures which represent
average conditions for the area, and all parts of the trees are sprayed to
or slightly beyond the point where run-off begins. Spraying is done by
personnel of the Division of Fruit Insect Investigations, and the man in
charge actively participates. Obviously, the risk of errors in mixing and
of anything less than thorough spraying (which might introduce extreme or
uncontrolled variation, thereby endangering the accuracy of all biological
and chemical data subsequently collected) is somewhat lessened when he does
his share of the spraying. He is then better able to interpret unexpected
developments, of which there are many.
To reduce the danger from complicating factors, such as apple scab
and blight, the prebloom and petal-fall sprays for disease control are also
applied by the laboratory personnel.
Method of Sampling and Handling the Apples
Sampling begins in the spring as soon as the fruit is large enough
to be handled. This is generally about 4 days after the first eggs have
hatched in the field, or between the first and second regular cover sprays.
Samples are taken from each plot usually 1 day before and 1 day after ap-
' plication of each of the seven or eight cover sprays, as well as on occa-
sional dates between sprays and at intervals after the last spray. In this
way information is obtained on changes in the effectiveness of the various
materials during the interspray periods, although the periods covered by the
records are usually 2 days shorter than the actual interspray periods.
Two trees in each plot are used to provide the samples, one of the
pair being dropped and-a new one substituted after each spray so that every
tree in the plot will be sampled during the course of the season. Each
sample for biological tests consists of 80 apples, 40 coming from each tree
of the pair, and is stratified, 30 percent (12 apples) coming from the top,
40 percent (16 apples) from the middle, and 30 percent (12 apples) from the
bottom portion of each tree. These samples are selected without respect to
the quantity or character of the spray deposit.
Since 1936 the samples taken for chemical analysis have been dis-
tributed in approximately the same ratio as those for biological tests, and
since 1937 a still closer relationship has been maintained by taking both
sets of samples from the same trees at the same time.
Apples for the biological tests are impaled on 4-inch wire pins through
the calyx end while the apple is grasped by the stem as shown in figure 1.
Some contact with the apple is unavoidable, but extreme care is taken to.
prevent rubbing or removing any deposit. The apple is removed with the stem
intact and the surface is not touched thereafter. The pinned apples are
then mounted in numbered holes on numbered sticks, each stick holding 10
apples. Each stick carries a stratified sample, with each part of both
trees represented, although of course not equally. This makes possible its
use as a replication, with much of the variation that might be due to
deposit differences, as between the three parts of the tree, equalized.
The samples for the various parts of the trees are always placed in the same
order on each stick, which permits separate analysis later, if desired.
Appropriate records are made on sheets previously prepared for each
treatment, and the sticks are then loaded into the apple cart, a specially
built trailer which can carry 120 sticks, making 15 samples of 80 apples
each (fig. 2). The canvas covers are tied down after the sampling is
completed, and the apples are hauled to the laboratory.
At the laboratory the apples sampled from each plot are kept together
and set up on a large rack in an unheated, dry basement room. There a ring
of sticky banding material is placed about the calyx of each apple close
to the pin to prevent subsequent escape of larvae down the pin or the
entrance of worms through the calyx, where an accurate count of numbers
would be impossible. The material used for this purpose is ordinary tree-
banding material which is thinned and poured (while hot) into lead-foil
tubes. These have 1-inch applicators on the end, which greatly facilitates
application of the material. Approximately one-half day is required for
two laborers to treat all the apples in a full-sized experiment.
Methods of Obtaining Newly Hatched Larvae
During the period of the experiments large numbers of moths are kept
emerging in the insectary so that there will be no lack of material. Eggs
are obtained by concentrating 200 to 303 adults each in mailing tubes 6
inches long and 3 inches in diameter, and holding them under condition
which stimulate them to oviposit on the inner walls of the tube, which have
been paraffined. When the eggs of each lot have developed to the point
where the most advanced are nearly ready to hatch, they are placed in a
refrigerator and held until needed. For the most efficient work, the eggs
must be sufficiently advanced in development to begin hatching within an
hour after removal from the refrigerator. They must not be allowed to get
too old, however, or larval vigor will be reduced.
Infesting the Fruit
The larvae are transferred to the fruit in an "infestary," a basement
room insulated and uniformly lighted (except at the transfer table). The
temperature of the room is maintained at 80 to 82 F. and humidity at 55 to
60 percent. The oviposition tubes are placed on a rack under a 300-watt
lamp. As the larvae hatch they quickly crawl out into a shell vial inserted
in a hole in the side of the oviposition tube and are picked up with a small
On the transfer table (fig. 3) are two spindles supporting removable
circular boards, with numbered holes around the circumference, each board
accommodating as many as 15 apples. These are used to hold one apple from
each plot as it is being infested, after which the apples are returned to
the sticks from which they were removed.
Since larvae hatching in different tubes may differ in vigor because
of differences in the age of the eggs, only two are placed on the apple at a
time. The board is revolved until each apple has received 8 larvae. Thus
the hatch in any one tube will be spread over more apples, and the larvae
will have a chance to scatter on the fruit, their attacks on one another
being thereby lessened.
As a rule the larvae are placed on the stem so as to minimize the
danger that poison will be picked up on the camel's-hair brush and trans-
ferred to larvae going on other apples.
When the infesting operation is in progress two men are employed for
the work. One reports at 6 a. m. and the other 2 hours later. The first
man immediately begins to remove the oviposition tubes from the refrigerator,
a few at a time, thereby ensuring a uniform rate of hatching, at least during
the early part of the day, since as a rule hatching begins within an hour
after removal of the tubes. With this procedure eggs are hatching over a
10-hour period. It usually takes from 2 to 2- days to infest a 15-treatment
experiment, which requires 9,600 larvae.
Between mid-May and September, 1938, 260,000 larvae were applied to
laboratory-field samples. More than a million have been used in the past
Determination of Results
After the apples are replaced on their original sticks they are
allowed to remain undisturbed for a few days; then the sticks are stored on
a stationary rack in one corner of the room until the apples can be examined.
Usually 8 to 10 days must intervene before the infestation is determined,
because some larvae may, after entering, remain immediately under the skin
for several days. The apples are cut open as shown in figure 4, and all worm
and sting injuries recorded separately. The injuries are scored as worm
entrances if they penetrate one-half inch or more or if the larvae are still
Larvicidal efficiency is calculated on the basis of successful worm
entrances in the treated apples as compared to those in the unsprayed check.
In unsprayed check samples 82 percent of the larvae, on an average, suc-
cessfully entered the fruit in 33 experiments completed in 1938.
Reliability of Experimental Procedure
The analysis of variance (6) is applied to all experiments and odds
of 99 to 1 are required for significance. The average difference in larvi-
cidal efficiency between treatments required to indicate significance has
been 7 percent. When during the course of a season a treatment runs con-
sistently worse or better than another, such a trend can usually be con-
sidered significant, even though some of the individual differences may not
be wide enough to give the required odds.
A recent analysis of the 86 regular experiments completed since 1936,
in which some 700,000 larvae were used, showed that in only 7 of the 86 did
the standard error exceed 10 percent of the mean. This occurred where the
mean number of worm entrances with the unsprayed sample included was less
than 15 percent. In experiments where about 25 percent of the worms entered
the fruit the standard error averaged only 7 percent of the mean, and where
half the worms entered it averaged only 4 percent of the mean.
The analysis of variance applied to the data for 1935 suggested that
if the number of worms per apple were increased from 6 to 8, and if the
apples from the two trees were mixed to make each stick truly representative
of the treatment, the sample could be reduced from 100 apples to 80 and at
the same time a smaller standard error would result.
Two trees per plot were adopted as standard after 1935, following
a survey of that year's data, when samples of 50 apples from each of two
trees per plot infested separately showed that significant differences at
odds of 19 to 1 existed in 12 percent of 175 pairs of trees tested. This
is slightly more than would occur in random sampling of homogeneous material.
It was concluded that by using two trees. as the source of the sample,
significant variations of the samples from the true plot mean would be rare,
and by using the trees in rotation all plots would be fairly represented
during the course of the season.
Type of Information Obtained
Illustrations of the type of information obtained and its applica-
tion are given in figures 5 to 8 inclusive. Figure 5 indicates the rela-
tive effectiveness, at intervals during the season, of the chief materials
Figure 6 illustrates the differences in efficiency that may exist at
different levels in the tree despite thorough spraying. The records for the
separate portions of the trees from 9 to 15 different spray treatments
tested in 1934, on 11 dates during the course of the season, showed that the
average efficiency for the entire season was 80 percent greater in the
lower parts than in the tops of the trees sampled. It was on the basis of
this information that the practice of stratifying the samples was adopted.
The principal reason for the difference in efficiency between the
deposits in the top and bottom of the tree is the fact that, regardless of
how uniform or thorough the spraying may be, the downward drip of spray
material that goes on for some time after spraying has ceased tends to build
up deposits in the lower part of the tree at the expense of those higher up.
There is also the tendency of rains to remove spray material from the top of
the tree and to redeposit some of it on fruit in the lower parts, as reported
by Marshall and Ford (3). Added to this is the greater exposure of upper
deposits to the action of rain and other weathering effects.
In figures 7 and 8 larvicidal efficiencies have been converted to
probits (1) and spray deposits to logarithms. Each point represents a
stratified sample of 80 apples infested with 640 larvae and a stratified
sample of 50 apples utilized for the chemical analysis. It is apparent from
figure 7 that deposits of lead arsenate in excess of the median lethal dosage
with weak (I-l--100) bordeaux mixture are less toxic when fresh than after
having been subjected to rainfall, and that there is very little correlation
between amount of fresh deposit and larvicidal efficiency. Reference to
figure 6 will show that from late in June to late in July, 1937, which was
a very dry period, the efficiency of lead arsenate was at a very low level
despite the application of the fifth and sixth cover sprays. This low
toxicity may account for the relative ineffectiveness of lead arsenate in
arid regions or in hot, dry seasons.
Reference to figure 8 will show that given deposits of phenothiazine
prior to June 15 were far less toxic than later in the season, and that the
material used in 1938 was less toxic than that used in 1937. The reasons
for these differences are not important to this discussion, but it is evi-
dent that the additional accumulation of paired data of this type will
eventually make it possible to determine more of the reasons for this lack
of correlation, and may possibly give leads as to methods for increasing
The study has also revealed the fact that the effectiveness of a
spray treatment cannot be estimated entirely by the quantity of spray
deposit present. Sampling errors cannot account for the poor fits of the
regression lines in figures 7 and 8. The wide scatter may be attributed
to the fact that the dosage measurement is not an exact measure of the
amount of poison ingested. It is merely an indication of the extent of the
poisonous barrier that is present. The character of this deposit thus
assumes considerable importance, for it apparently determines how much of
the load will be ingested.
Disadvantages of the Laboratory-field Method
as Compared to Large-scale Tests
1. The method does not measure the end result. By end result is
meant the proportion or amount of injured fruit as determined by preharvest
and harvest counts of dropped and picked fruit.
2. It does not show the effect on larvae of spray deposits on foliage
and in the calyx end. This is a serious disadvantage early in the season,
when larval activity is retarded and when poison picked up on the foliage
may have more time to weaken the larvae sufficiently to prevent entrance.
Practically 94 percent of all eggs are deposited on the foliage, according
to extensive counts made in 1938 at Vincennes by S. A. Summerland and others
of the Vincennes staff. Analyses by the Division of Insecticide Investiga-
tions show that deposits on the foliage are much heavier than those on the
fruit. The actual effectiveness of certain treatments will thus be under-
estimated, that of lead arsenate possibly more than others.
3. It does not show the toxicity of sprays to moths. In small plots
this effect does not register in the final results either. It was found in
1938 that certain fixed nicotine-oil sprays killed up to 50 percent of the
moths in the tree at the time of spraying, whereas lead arsenate killed
less than 1 percent. (Spraying must be very thorough and must be done
quickly to obtain a high kill of moths with nicotine.) The work by Hough
(2), in particular, demonstrates what can be accomplished by certain more
active combinations designed to liberate free nicotine.
4. It does not show the ovicidal effect, which may be as high as a
90 to 95 percent kill of the eggs present at the time of spraying. Usually,
however, the so-called ovicides as used by the grower kill a much lower
5. It does not include the effect of spray deposits on the retention
of eggs on foliage. A tendency has been found on the part of treatments such
as tank-mix nicotine-bentonite-soybean oil to cause eggs to slough off more
rapidly, particularly from the upper, more exposed surface of the foliage
and in the tops of the trees.
6. It does not measure the repellent or attractive effect on gravid
females. Recent observations at the Vincennes, Ind., laboratory have indi-
cated that spray deposits vary considerably in their repellency or attrac-
tiveness to moths. In a large area such influences might be nullified by
inability of moths to escape them, but in small-plot work it is obvious
that such differences could easily modify the results.
7. It does not -include the effects of sprays on larvae already in
the apple. A recent laboratory test showed that up to 40 percent of 1-day-
old larvae and 15 percent of 4-day-old larvae can be killed in the apple
by certain nicotine-bentonite oil sprays.
8. It does not show the effect of the treatments on parasitic and
predatory insects, which observations at the Vincennes laboratory have shown
to vary greatly.
9. Other projects must of necessity be secondary, since everything
must be efficiently synchronized with codling moth activity or the work will
bog down. Codling moth development sets the pace by regulating the fre-
quency of spray applications and sampling, and the laboratory end must be
kept up with field developments. Consequently some reserve help must be
available which can be shifted from other projects when needed.
Some of these deficiencies in the method are also characteristic of
the usual type of field-plot work. The effects of several of the factors
can be measured and means of evaluating the others are gradually being
Advantages of the Method
1. The method avoids the extremely important influence of interplot
migration of moths. It is also entirely independent of the native moth
population. Too often definite conclusions cannot be drawn from regular
field tests when weather conditions combine to prevent a sufficiently high
2. It makes possible a study of the relative effects of rains, winds,
high temperatures, humidity, length of spray interval, and frequency and
number of sprays on the effectiveness of different treatments at any and
all times during the season. When there is added to this the chemical
analysis of spray deposits and measurements of growth, the reasons for the
effects attributed to the factors named can be determined more accurately.
3. The method tells more about a treatment in less time than any
other. Weak and strong points can be located. The results of efforts to
correct weak points can be determined quickly. For example, in 1935, within
3 weeks after the use of phenothiazine was begun in the field, it was de-
termined that this material is too susceptible to rain weathering. Larvi-
cidal data supported visual observations that rains, wind, and heavy dews
removed deposits rapidly. The adverse effects of two copper fungicides on
tank-mix nicotine-bentonite were noted early enough in 1938 to make possible
the inclusion of three more copper compounds in the test that season. Tests
of the usual type would not have shown that the deposits were less resistant
to rainfall and less toxic. When all five compounds gave the same results
it became evident that more effort should be expended on overcoming their
effect on the insecticide rather than continuing to search for safer copper
materials among the many now on the market.
4. When accompanied by chemical analyses, the point of diminishing
returns in a spray program is indicated. Thus the proper interval for
spray applications and the proper dosage per application can be intelli-
gently adjusted to compensate for growth and weathering with greater chances
of avoiding the use of excessive quantities of material when not needed and
when they may cause injury. For example, the tank-mix nicotine-bentonite
dosage was cut to two-thirds or one-half strength in second-brood sprays
beginning in 1936, after the tests of 1935 had indicated that weaker mixtures
timed the same as lead arsenate would maintain a highly efficient deposit
after such a deposit had once been built up early in the season.
5. The method is flexible. Injurious treatments can be dropped and
others substituted, either in the same set-up or elsewhere at almost any
time. New leads or approaches to the problem, which appear much more often
with this method than in ordinary field-plot experiments, can thus often be
followed the same season.
6. The method weeds out injurious treatments or ineffective treat-
ments before time and money have been wasted in large-scale tests. It is
highly dangerous to put any new treatment to large-scale use without prior
small-scale all-season tests, since injury often does not occur until
several spray applications have been made. The method makes it possible to
improve and develop a new combination until its weak and strong points are
thoroughly understood, and to narrow the field to one or two such treatments
which can be compared with the standard lead arsenate treatment on a large
enough scale to offset the effects of migration, and to avoid the many
other disadvantages inherent in small-plot work.
7. The method forces a closer observation of the effects of the
several treatments throughout the season, on the tree, on diseases, and on
other insects, largely because of the necessity for climbing over the trees
before and after every spray to take the samples.
8. Seasonal conditions affect the results only through the influence
they exert on the character and amount of spray deposit. Thus the effects
of weather on the spray treatment can be separated from its effect on the
9. The method makes it possible to study variability in effectiveness
of deposits within the tree, which was found to be greater than between
trees. The efficiency of the various treatments in tops and bottoms of the
trees and the change in their relationship after each spray can be deter-
mined. (See fig. 6.)
10. Although the method underestimates the mortality that would
occur under normal field conditions, particularly early in the season, it
closely approximates the kill that could be expected of larvae hatching on
or very near to the apple under conditions favorable to the insect, when
good control is usually extremely difficult to obtain. If proper allowance
is made for defects or weaknesses in the method when evaluating different
- 10 -
treatments, the odds are great that the expected results can be obtained in
large-scale field tests. This is indicated by the results obtained in
1937 (7) with the tank-mix nicotine-bentonite-soybean oil combination,
developed by this method, and by its further successful use on 737 acres in
southern Indiana by five growers in 1938.
(1) Bliss, C. I. 1935. The calculation of the dosage-mortality curve.
Ann. of Appl. Biology 22: 134-167, illus.
(2) Hough, W. S. 1938. The use of nicotine in codling moth control with
special reference to its effectiveness in killing moths. Jour.
Econ. Ent. 31: 216-221, illus.
(3) Marshall, G. Edw., and Ford, 0. W. 1933. The relation of apple spray
schedules to the arsenious oxide and lead residues. Purdue Univ.
Agr. Expt. Sta. Bull. 381. 19 pp., illus.
(4) Lathrop, F. H., and Sazama, R. F. 1932. A laboratory-field method for
the study of the efficiency of codling moth sprays. Jour. Econ. Ent.
25: 83-96, illus.
(5) Siegler, E. H., and Munger, Francis. 1933. A field and laboratory
technique for toxicological studies of the codling moth. Jour. Econ.
Ent. 26: 438-445, illus.
(6) Snedecor, George W. 1934. Calculation and interpretation of analysis
of variance and covariance. 96 pp. Collegiate Press, Inc. Ames,
(7) Steiner, L. F., and Sazama, R. F. 1938. Experiments with tank-mix
nicotine-bentonite-soybean oil for codling moth control. Jour.
Econ. Ent. 31: 366-374, illus.
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Figure 1.- .
with t1. -
is _a 7 31 on a Nire pin and removed from the tree
4. .. 9
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Figure 2.-The .
of 2 tree;,
1,200 ut -
..v:n, of 80 apples, taken from the 3 portions
:,' .. 8 numbered sticks and loaded into a
o Th -railer holds 120 sticks, or 15 samples-
Figure 3.-One apple from each treatment is placed on a circular board,
and 8 newly hatched larvae taken from the vials in the mailing-tube
oviposition cages are placed on each apple, after which it is re-
turned to its original stick.
Figure 4.-After the larvae have entered the fruit it is concentrated
on a separate rack and held for 8 days before being examined. The
"worm" and "sting" injuries are recorded separately for each apple
after it is cut open.
LARVICIDAL EFFICIENCY IN UPPER
VS. LOWER PARTS OF TREES
LOWER fit -%
I I K, ", ", \
,- .. ".,
". ;UPPER 'I
Figure 5.--Larvicidal-efficiency curves for representative spray
treatments during the 1938- codling moth season, with dates of
spray applications and rainfall indicated. Lead arsenate was
applied on the same schedule as the nicotine treatments. Har-
vest began August 31. UIBRARY
STATE PLANT BOA R.
* *5 I
COVER SPRAYS OF
AFTER JULY 7
- TANK MIX NICOTINE BENTONITE
-------PROCESSED NICOTINE BENTONITE
WITH MINERAL OIL
Figure 6.-Percentage larvicidal efficiencies of deposits in the tops
as compared with those in the lower parts of 21-year-old Jonathan
trees, some sprayed with the lead arsenate program recommended in
Indiana and Illinois and others with a tank-mix of nicotine sul-
fate-bentonite and soybean oil during the season of 1937.
LEAD ARSENATE- BORDEAUX
AS203 DEPOSITS IN LOGARITHMS
6.0o 7.0 8o:0 9.0 io
AS203 DEPOSITS IN MMG. PER S
Q. CM. OF
180 20.0 22.0
Figure 7.--Efficiency-deposit regression lines for fresh and weathered
deposits of lead arsenate plus 3/4-1-lOO1 bordeaux mixture after
June 15, in 1936, 1937, and 1938.
7.5 LATE 1937 0
99 LATE 1938- +
98 EARLY IN SEASON +
90. + +
Z 6.0 0
S80 ^ +
U- + 4
1I I 9 01-, + .
l40- 0 "
0.40 0.50 0.60 WO7 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60
PHENOTlJIAZINE DEPOSITS IN' LOGARITHMS is __ _02_3 ,
3:0 4.0 5.0 6.0 8.0 100 150 2.0 250 360 4d0
DEPOSITS IN MMG. PER SQ. CM.
Figure 8.--Efficiency-deposit regression lines for phenothiazine ap-
plied after June 15 in 1937 and 1938 and before June 15 in both
UNIVERSITY OF FLORIDA
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