P LANT BOARP
July 1948 ET-256
thiited States Department of Agriculture
Agricultural Research Administration
Bureau of Entomology and Plant Quarantine
STAINING METHODS FOR DETECTING WEEVIL INFESTATION IN GRAIN
By J. C. Frankenfeld
Division of Cereal and Forage Insect Investigations
The eggs and immature stages of the rice weevil (Sitophilus S (Lo)
and the granary weevil (So gEranriua (L* )), hidden wi 'in he of
wheat, corn, or other grain, are invisible to the naked eye and cannot be
detected by ordinary inspection methods. Grains that grade No. 1 my
actually have a high percentage of infested kernels* If this grain is
used for milling, the different stages of the weevils, together with the
cast skins and excreant, will be ground up in the milling process and
the finished flour or meal will thus be contaminated with the fragments*
In order that the miller may produce a pure product he must know the
condition of the grain he is grinding. So far as the miller is concerned,
it matters little whether the infestation in the grain is alive or dead,
or what stages are presents since any stages of the weevils from egg to
adult, whether living or dead, will contaminate the finished product. A
miller may therefore buy a No. 1 grade grain, which apparently is free of
infestation because of fumigation and cleaning methods applied prior to
its arrival at his mill, but which may, nevertheless, be heavily infested
with immature stages of weevil hidden within the kernels.
A simple, practical, and inexpensive method has been devised by the
author for detecting this hidden infestation in grain. It consists of
treating samples of the grain with a dye which stains the egg-plugs and
weevil punctures. In depositing her egg the female weevil first drills
a tiny cavity into the surface of the grain kernel. After depositing
her egg in this cavity she seals the opening with a gelatinous secretion,
which hardens and serves as a protective plug* This plug remains on the
grain throughout the development of the immature stages, and, unless
removed by the emerging adult weevil, will remain on the infested grain
indefinitely. Therefore, no matter what stages of the weevils may be
present, any infested kernel can be easily detected by the use of a dye
that has an affinity for the egg-plugs.
There are two dyes which may be used in testing for weevil infes-
tation in grain. Because of its decided advantages, the author prefers
the aoid faohsin dye. It is prepared with the following ingredients,
Acid fuchain 0.5 gmi.
Glacial acetic acid 50.0 cc.
Distilled water 950.0 co.
ix the distilled water and glacial acetic acid and then add the acid
fuchuin which dissolves readily in this solution. Care should be taken
iJUL 2 2 1948
not to confuse acid fuch3in with basic fuchain, which is entirely
different in its action and is not suitable for detecting weevil infes-
tation in grain. Acid fuchsin and glacial acetic acid are relatively
inexpensive and may be obtained from any drug or hemical supply house.
It is not necessary to prepare fresh solutions of acid fuhsino
Any desired quantity may be prepared at one time, stored, and used as
needed without loss in efficiency. The same solution may also be used
a number of times without losing its efficiency. If after prolonged
usage the solution tends to become murky it should be discarded.
The samples of grain to be tested should be placed in a suitable
container, preferably a shallow dish large enough to adequately hold the
sample and solution. Enough of the solution should be used to cover the
kernels completely. The grain is left in this solution from 2 to 5
minutes. If left in the solution for a longer period the kernels will
absorb enough of the solution to color them slightly, thus making it more
difficult to locate the egg-plugs. If the grain is permitted to soak in
warm water for 5 minutes before being treated, less staining of the
kernels results without impairing the staining of the egg-plug. The
water should be poured off before the fuohsin solution is added. After
the grain has been immersed in the fuchain dye from 2 to 5 minutes pour
off the dye and wash the grain in tap water until all the excess dye has
been removed. It may then be examined under water or drained and spread
on a glass plate or similar surface for examination.
The acid fuohsin stains the gelatinous egg-plugs a deep cherry red,
whereas feeding punctures and mechanical injury are stained a light pink.
The egg-plugs (fig.l) are about the size of an ordinary pin prick and are
readily seen with the naked eye, but the use of a reading glass is helpful
in finding them.
Besides staining the weevil egg-plugs, this dye will also stein
weevil feeding injury, as well as entrance and exit holes of the lesser
grain borer. These are stained a lighter color, however, and can therefore
be easily differentiated from weevil egg-plugs. Mechanical injuries, as
a result of threshing and subsequent handling of the grain, will also be
stained. However, they may be easily distinguished from the egg-plugs by
their shape, size, and lighter color. Insect feeding injuries are round
and smooth in outline, while mechanical injuries are very irregular. Grain
that has passed through cleaning machinery, such as scourers and disk
separators, prior to milling, frequently bears injuries that greatly
resemble the feeding injuries of weevils and grain borers. It is practically
impossible to differentiate these two types of injury. However, the miller
who wants to know whether the grain contains weevil infestation can find
this out by using the stain on samples of the grain before it is run through
the cleaning machinery, thus avoiding any confusion in distinguishing
weevil egg-plugs and weevil or grain borer feeding injuries from mechanical
The proportion of weevil egg-plugs to feeding punctures will depend
largely upon (1) the number of adults to which the grain has been exposed;
and (2) the condition of the grain, particularly as to moisture content.
Apparently the adult weevils feed on the endosperm of the grain kernel
in the process of drilling the cavities in which they lay their eggs.
Thus, unless the weevils are disturbed, eggs are usually deposited in
all cavities that the adults drill into the kernels. If the populations
are very large many punctures that do not contain eggs may be found.
This is because the female weevil was disturbed while drillixxg the egg
cavity and did not return to deposit the egg. The author does not con-
sider this a significant factor, however, for in all such oases en-
countered there were one or more egg-plugs in addition to the egg-free
If the moisture content of the grain is comparatively low, that is
11 percent or less, the female weevil may drill a cavity but, finding
conditions unsuitable, will not deposit an egg. Such grain will generally
have a comparatively high percentage of egg-free punctures. In numerous
dissections of kernels that had been treated with this dye to locate the
egg-plugs, weevil eggs were found under every plug. The female weevil
does not seal the cavity in the kernel unless she has deposited an egg.
By actual count the total hatch is from 85 to 90 percent of all eggs laid.
With moisture and temperature conditions remaining favorable, all larvae
that hatch will complete their development.
Since this dye stains the weevil egg-plug, it is suitable for determi-
ning weevil infestation in wheat, corn, and sorghum grains. Fifteen va-
rieties of hard and soft wheat have been tested, and on all of them the
egg-plugs could be readily detected. Polished rice takes up too much
stain to reveal egg-puncture plugs. The stain has not been tried on other
grains, and its effect on the eggs of weevils that glue their eggs to the
surfaces of dry beans and peas has not been determined.
Weevil eggs may be readily distinguished from the eggs of other
species of grain-infesting insects, because all other species lay their
eggs loose among the kernels, in larger feeding cavities in the kernels
or in floury material mixed with the grain.
U. S. Bureau of Entomology and Plant Quarantine ET-256
The fact that this is a repo -t of a study made under the Research
and Marketing Act of 1946 was inadvertently omitted.
Page 3, -last paragraph--In place of first 8 lines, including
that which 6nds "This dye, commonly used as a," substitute the
The other preparation that may be used is an
iodine solution which is prepared by diluting 20 mi.
of Lugol's solution, obtainL.bl2 from- --ny d rug or
chemical supply house, with atcr to make a total
volurne of 900 ml. ThIs preparation, commonly used \
In general, the method used is the same as for the fuchain dye
except that a 10-minute period is required to obtain maximum staining.
An iodine solution should always be fresh when used because it quickly
loses its efficiency upon standing. It is also important to examine the
grain soon after staining because the color gradually decreases in
intensity when exposed to the air.
Figure l.-Wheat kernels showing egg-plugs of the granary weevil stained
with acid fuchsin.
UNIVERSITY OF FLORIDA
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