A technique for the recovery of very small dead insects in mortality experiments


Material Information

A technique for the recovery of very small dead insects in mortality experiments
Physical Description:
Lawson, F. R
United States -- Bureau of Entomology and Plant Quarantine
U.S. Department of Agriculture, Bureau of Entomology and Plant Quarantine (Washington, D.C )
Publication Date:

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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 30345587
oclc - 781638779
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Full Text
ET-158 Apri 1 1940

United States Department of Agriculture
Bureau of Entomology and Plant Quarantine


By F. R. Lawson, Division of Truck Crop and Garden
Insect Investigations

The apparatus and methods described were developed in con-
nection with studies of the resistance of sugar beets to the beet
leafhopper. In these experiments a given number of females were
allowed to oviposit in a single leaf for a given length of time.
For the greatest efficiency in experimentation, it was necessary to
determine accurately the total hatch of nymphs and the mortality of
these nymphs from the first instar to maturity. This required not
only the recovery of live insects at the end of the experiment but
also all dead individuals. Some of the latter died in the first
instar, and leafhoppers which die in this stage shrivel to a mere
speck, impossible to find in any ordinary cage by the usual methods.
All known types of leaf cages small enough to permit recovery
of dead leafhoppers cause death of the leaf after a few days because
they depend on pressure for attachment to the leaf. This is a
serious disadvantage in long experiments.

Drawings and dimensions of the cage that was developed to
overcome these difficulties are shown in figure 1. The top and
bottom are made of fine-meshed muslin cloth, the sides and ends of
celluloid. There is a round hole 7/16 inch in diameter in one end
of the cage and another hole 13/16 inch in diameter in the other
end. Figure 2 shows construction details of a wire support for the
cage. The method of mounting the cage on potted beet plants is
illustrated in figure 3.

The first step in mounting is the wrapping of a band of cot-
ton around the leaf petiole to form the cotton plug. Unwrapping of
the band is prevented by twisting a few strands together with the
fingers. The cotton should be a good grade of batting of the more
springy type, that is, having stiff fibers. The common absorbent
cotton tends to lose its resilience if it becomes wet when the plants
are watered. Enough cotton should be used to close firmly the
opening around the petiole but not enough to cause more pressure
than is necessary.


After the cotton, the wire support is put in place by pushing
the ends of the wire into the soil. Then the leaf, which is usually
wider than the large hole in the cage, is gently folded or rolled,
and the cage is pushed over it and down around the cotton plug.
A cork stopper closes the small upper hole in the cage.

If the bottom of the cage rests on the damp soil of the pot,
it is well to put a small piece of wood under it as a support.
Otherwise the cloth will rot in a short time.

Insects can be put into the cage or removed from it with a
glass pipette through the small upper opening. If the pipette is
only slightly smaller than the hole in the cage, insects cannot
escape past it during these manipulations.

At the end of the experiment living insects are removed and
counted. The petiole can be cut below the cotton plug to facili-
tate handling and permit shaking of the cage to make sure that all
live insects are taken out. Dead insects are then removed with the
apparatus diagramed in figure 4. The rubber tube shown on the right
of the figure is attached to a machine that produces a strong suc-
tion. The long glass tube at the top of the figure is inserted into
the cage and the cage is carefully vacuum cleaned. The cotton plug
and the leaf are usually removed over a piece of paper and cleaned
outside the cage. All dead insects, cast skins, and dirt are de-
posited on the cloth filter. This filter is then removed and the
insects are sorted and counted under the microscope.

The data at the end of the experiment include total live
insects and total dead in each instar. From these data the total
hatch and the percentage of mortality are calculated.

Various tests have shown this technique to give highly ac-
curate results provided a strong enough suction is used to pick up
all insects and provided the suction tubes, cage, and leaf are dry.

In some experiments the leaf surface becomes very sticky
from leafhopper excrement or the exudation of sap from feeding
punctures. Dead leafhoppers may become stuck so tightly that they
are not picked up by the suction machine. This can be prevented by
soaking the cages with their contained leaves and insects in water
for about an hour and then drying. The sticky substance is dissolved.

After live insects are removed from the cages, if the clean-
ing process is delayed as may be necessary in large experiments, the
leaves may become so dry and brittle that they break into small
pieces when cleaned. This debris is picked up by the suction
machine and mixed with the dead insects, which makes sorting a very


laborious job. This can be avoided by placing the cages in a moist
relaxing jar for an hour or more before cleaning. If they are left
in the jar more than a few hours, a spoonful of formaldehyde should
be added to the water to prevent molding.

If the cages become dirty, they can be washed in water and
scrubbed inside with a bottle brush. With continued use the cages
may become warped and misshapen. Most of this deformity can be
corrected by drying them, after washing, on the spreader shown in
figure 5. The spreader is inserted into the cage by pressing the
two short arms together until the long arms can pass through the
large hole in the cage. The spring is then released, allowing the
long arms to press outward on the celluloid sides of the cage,
forcing it into approximately its original shape.

Method of Constructing the Cage

First prepare a wooden form. Using a coping saw, cut from a
1-inch board a plug the size and shape of the cloth top of the cage.
Enlarge the saw cut so that one thickness of cloth and celluloid can
be held tightly between the plug and the form when the plug is in
the hole from which it was cut.

Using the wooden plug as a pattern, mark the cloth with a
soft pencil and cut 1/8 inch larger than the pattern on each side.
If it is desired to number the cages, a numbering machine that
stamps consecutive figures can be used to print numbers on the cloth.

The celluloid is cut into strips 1-1/2 inches wide and 19
inches long. The holes are cut by clamping one or more pieces of
celluloid between wooden blocks and boring through with a carpen-
ter's bit.

To assemble the cage, place the wooden form flat on the
table with the plug out. Put one of the pieces of cloth, which are
to form the sides of the cage, flat in the bottom of the hole
with the edges turned up evenly along the sides of the form. Then
place the celluloid strip on edge around the inside of the form
and inside the turned-up edges of the cloth. Now force the wooden
plug into place inside the celluloid. Cloth and celluloid will
now be held firmly in place. Cement the overlapping ends of the
celluloid strip together with acetone. Turn the form over and
cement the cloth to the celluloid by allowing a little acetone to
run down the crack between the wooden plug and the form.

Remove the wooden plug and the partly completed cage from
the form and place the second piece of cloth in the bottom of the
form as before. Fold the cage slightly and put the unfinished side


down in the hole in the form. It will now expand sufficiently to
hold the cloth in place without the wooden plug. Turn the form over
and cement the cloth to the celluloid. Remove the cage from the form
and stick down the cloth in any place where it is not firmly at-
tached. If the cloth does not lap over the celluloid more than
1/8 inch, any wrinkles along the edge that is cemented to the cel-
luloid can be worked out by moistening the cloth with acetone and
smoothing with the fingers.

Construction of the Suction Apparatus

All the connections of this apparatus (fig. 4) must be
made airtight. The large rubber stopper, which is set into the
wooden block, should be firmly cemented to the block. The curved
end of the long glass tube greatly facilitates cleaning cages, since
corners and the less accessible parts of the cage can be reached
with it. The glass tube that fits into the large rubber stopper
and holds the cloth filter in place is made larger than the other
tubes to provide a larger area of filter. As a result of this
construction, a strong current of air passes through the small
tubes, preventing insects from becoming lodged, and a weaker current
passes through the filter without forcing the insects through
the cloth.

The Effect of the Cage on Physical Factors

When these cages were put on beet leaves in the greenhouse,
the temperature inside the cages was usually lower than outside.
This difference increased as the temperature outside became higher.
For instance, at 800 F. the difference was approximately 20; at
900 the difference was about 5 degrees. When outside air was forced
into the cage through a tube, the difference was still greater,
being under our conditions 40 at 800 and 9.50 at 900. This sug-
gests that these temperature differences are due to evaporation
from the leaf, since a current of air in the cage would increase

No measurements of light have been taken inside the cages,
Lut these cages appear to be well lighted. Some beet leaves inside
the cages have survived as long as 8 weeks in excellent condition.
Other leaves have turned yellow and died within 2 or 3 weeks under
conditions where they would normally have lived a much longer time
outside. Lower light intensities inside the cages probably are a
contributing cause of this abnormal behavior.


3 13


Figure 1.-The cage waed. Stippled parts of the drawing represent
close-meshed muslin cloth.


3 *



Figur 2.-Wire frame used to support the cage. The dimensions
need not be exact.



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FlIgre 4.--Diagramatic cross section of appratus used In roving
dead leafhoppers trom the c The arrows Indicate direction
of air current.



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.I~t.PU aed oditd.
esadas era.


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