Note: Descriptions are shown in the official language in which they were submitted.
.3~ :
The present invention relates to a new anti-hemocytic
~rum for suppressing the imrnunologic mechanism of insects and
a method for the preparation thereof. The new serum finds ~ ~;
application in pest control of such insects as the cabbage
white butterfly, the bee moth, and the Colorado potato bettle. ~
Various biological preparations are ~no~,in for '
~, .
protecting plants from pests, for example preparationssupplied
under tlle trade ~ari~ Agritlol, Bact~n an~ ~isopore 28~. However,
pests soon acqulre i~nunity to these ~reparations, and with pro-
-~ 10 longed application they do not produce a positive effect.
According to the present invention there is provided
an anit-hemocytic serum for suppressing the immunologic mechanism
of insects which is inactivated plasma of the blood of immunized
animals obtained by the immunization of the animals with a
, . . .
suspension of hemocytes isolated from the cells of the bodies of ;~
the insects; the titre of hemocytoaggulutinis of the anti-
hemocytic serum being 1:256 to 1:2048.
Unlike conventional biological means of pest control the
serum suppresses the-i~nunological reactivity of insect pests by
retard~ing the-synthesis of antibodies. The advantages of the
serum include the destruotion of insects by suppressing their
immunity, the weakening of resistance of insects to bacteria,
and the harmlessness of the serum of the environment.
The serum can be used for controlling any species of
insect pests, and for every species of insects a serum is used
which has been prepared on the basis of cells of the body of ~;
an insect of the respective species.
According to the invention, the method for the prepara-
tion of the anti-hemoctyic serum for suppressing the immunolo-
gic mechanism of insects comprises removing the alimentary tractfrom the insects and the hemolymph, preserving the remaining
cells of the bodies of insects with a preservative and homo-
., ,. . .,.,, , .~
~c~
genizing them to form a suspension of hemoctyes, washing off thepreservative from the suspension. before lmmunization, and
immunizing animals four-times at intervals of five day~ by .~
injecting a 50~ suspension of hemoctyes in an amount of 0.07 ~`
:~. ml per 100 g of the weight of an animal, removing the whole blood ~ :
of animals after immunization, isolating the plasma of the blood
~:. and inactivating the blood.
The suspension of hemocytes is preferably preserved
by adding a mixture of the following components, in percent
. 10 by weight: -
sodium citrate . 3.5
glucose 2.0
chloramphenicol 0.015
distilled water up to 100,
and then a solution of 0.24 M of saccharose, 0.004 M of ethylene
diamine tetra-acetic acid, 0.01 M of sodium phosphate per 1000
. ml of distilled water having a pH of 7.0, and glycerin at a ~:
weight ratio of 1:2:1 respectively, is added to the resultant
mixture which is kept at a temperature of~ -20C to -2C.
~,
- The:method for the preparation.of the anti-hemoctylc ;
serum is effected in the following manner. -
Pest insects may be of different species such as
Galleria mollonella L, and Pieris brassicae L. The alimentary
:
tract of insects is removed to prevent the action of enzymes ~ ;
and the hemolymph is also removed. The remaining cells of the
bodies of insects are preserved in a..suitable preservative and
homogenized to form a suspension of hemoctyes. The following ;~
composition is used as a preservative ensuring the prevention of :
agglutination, melinization and oxidation, in percent by weight:
30 sodium citrate 3-5 ~ ~
glucose 2.0 . ~;
chloramphenicol 0.015 :
distilled water up to 10
- 2 - :
: ~ ., . ~.
To keep the hemoctye suspension at a temperature of ,~-
-~0C to -2C and to preserve the integrity of cells a solution
of 0.24 M of saccharose, 0.004 M of ethylene diamine tetra-
acetic acid, 0.01 M of sodium phosphate per 1000 ml of distilled
water being a pH of 7.0, and glycerin in a ratio of 1:2:1
respectively, is added to the resultant solution. Immunization '
~ of animals is carried out with the produced hemocyte suspension.
- For this purpose the hemocyte suspension is washed beforehand ~ -
` with a physiological solution to remove the preservative.
~` 10 Immunization of animals is then carried out four times at inter~
vals of 5 days by injecting a 50% hemocyte suspension in an
amount of 0.07 ml per 100 g of the weight of the animal. After `-
immuni~ation the whole blood of animals is removed, the plasma `
of the blood is separated and inactivated by keeping it at 56C
for 30 minutes. The resultant anti-hemocytic serum has a titre
of hemocytoagglutinins of 1:256 to 1:2048.
The anti-hemocytic serum wàs tested on different `
species of insercts. It was applied by spraying. The test results
showed a high activity of the serum. The death rate of insects
of the first generation was from 35 to 75% of the second genera~
tion from 85 to 100%, depending on the dose of the serum applied
to insects. The serum may also be used in com~ination with
entomopathogenic bacteria. ; `~
The present invention will be further illustrated by
way of the following Examples in which methods for the preparation
of the anti-hemocytic serum and methods for testing its activity
are described.
Example 1
2000 Caterpillars of Galleria mellonella L in their
dèvelopment period (nistars V and VI) are disinfected with !
ethyl alcohol and left for 5 to 6 hours for cleaning the alimentary
tract. The live caterpillars are placed in a preservative
- 3 -
consisting of the following components, in grams; sodium citrate
3.5; glucose 2.0; cloramphenicol O.OlS; disti]led water up to 100.
The preservative is used in an amount of 1 ml per 10 caterpillars.
Then one segment is removed from the front portion and one
segment from the rear por~ion of the bodies of caterpillars. The
alimentary tract is removed to prevent enzymatic action. The
residual bodies of the caterpillars are ground and placed to-
gether with said perservative in a magnetic mixer for 7 to 10
minutes for the complete separation of hemolymph. The resultant
homogenate is filtered and~centrifuged 6 to 7 times for 15
minutes at 1000 r.p.m. The centrifugate is left for 30 minutes
at room temperature and the upper fat-like layer is removed with
a cottonwool wad. The plasma of the hemolymph is poured o~f
and a solution consisting of 0.24 M of saccharose, 0.004 M of `~
ethylene diamine tetra-acetic acid, 0.01 M of sodium phosphate
per 1000 ml of distilled water having a pH of 7.0, and glycerin ~ ~-
in a weight ratio of 1 2:1 respectively, is poured onto the cells.
The resultant hemocyte suspension is kept until immunization of
animals at a temperature of -20C ~ 20C. Prior to immunization
the hemocyte suspension is washed with a physiological solution
to remove the preservative. Immunization of 12 300 g white
rats is carried out 4 times at intervals of 5 days. Each time
0.2 ml of a 50% hemocyte solution is injected. The first
immunizatlon is done subcutaneously, injecting 0.2 ml of a 50%
hemocyte solution into each rat. The subsequent immuniæations
are done intramuscularly and each time 0.2 ml of a S0~ hemocyte
solution is injected in mixture wïth 16,666 units of penicillin
to prevent possible infection.
Blood samples are taken from rats prior to the first
immunization to determine the presence of natural antibodies
in them against hemocytes of caterpillars. Seven days after the
last immunization the rats are dehematized. The separated blood
- 4 -
is placed in glass cy]inders and left at room temperature for
24 hours. The settled plasma of the blood ls poured off and
inactivated at 56C for 30 minutes. The anti-hemocytic serum
is produced in an amount of 15 ml. The titre of hemocytoagglu~
tinins is 1:2048.
The antl-hemocytic serum was tested on caterpillars of
Galleria mellonella L in their development period (II-IV and
.
V-VI nistars). Caterpillars were sprayed with the anti-hemocytic
serum in doses of 0.25 to 2.5 microliters for each caterpillar. `~
The effect of the anti-hemocytic serum on the immunologic
;~
reactivity of the insects organisms was checked every 6 hours~
At the same time the anti-hemocytic serum was tested in com-
bination with entomopathogenic bacteria Bac. Thuringiensis cereus
var. galleriae. The anti-hemocytic serum was used in doses of
from 0.25 to 2.5 microliters for each caterpillar. Bac. ~ ~
thuringiensis cereus var. galleriae was used as a 0.1% water sus- ~ `'
pension in treating caterpillars of the first generation and as
a 0.01% water suspension in treating caterpillars of the second
generation.
, ~
A control experiment (without treating caterpillars) and
treatment of caterpillars with heterogenous proteins and
entomopathogenic bacteria Bac. thuringiensis cereus var. galleriae
were carried out to compare the activity.
The effect on the viability of caterpillars was checked ~;
every 6 hours from the beginning of the experiments.
The maximum intensity of destruction of caterpillars
~,
Galleria mellonella L was achieved in the period from 12 to
18 hours from the beginning of the experiment.
The test results are given in Table~ 1 and 2.
.
~ 5 -
~ ~h33~
:
Suppresslon of Immunologic Mechanism of
the Effect of Bac. thuringiensis cereus ~;
Galleria mellonella
Types of experiments _ Effectiveness of
Destruction of Destruction of
caterpillars in caterpillars
less than 24 hours after 24
Nistar II _Nistar IV Nistar II Nistar IV
1. Check experiment -
(without treating
caterpillars) 0 0 0
2. Treatment with he-
terogeneous pro- ~
teins 0 0 1 1 ~;
3. Treatment with en-
tomopathogenic bac-
teria Bac. thuringi-
ensls cereus var.
g leriae 0 0 16 16
4. Treatment wi~h anti-
hemocytic serum32 32 32 32
5. Treatment with anti- ;~
hemocytic serum in
combination with Bac. !:
thuringiensis cereus~
var. galleriae 75 75 75 75
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Comparing the data given in the Tables it can be seen
that in treating caterpillars G mellonella L with the anti -
hemocytic serum their death rate in the second generation reaches
100% whereas due to -the use of entomopathogenic bacteria in the
second generation the efficiency of destruction of caterpillars
diminishessince they acquire immunity against ~hese bacteria.
Example 2
Caterpillars of the cabbage white butterfly Pieris
brassicae L are used. The process of preparation of the anti-
hemocytic serum is identical with that of Example 1.
The tests of the anti-hemocytic serum produced are
identical with those of Example 1.
Caterpillars of Pieris brassicae L were treated with
the anti-hemocytic serum in doses of 0.25 microliters to 4.5
microliters as well as with the anti-hemocytic serum in com-
bination with entomopathogenic bacteria Bac. thuringiensis cereus
var. galleriae which were used as a 0.1~ water suspension in
doses of 0.016 ml of each caterpillar~ The test results are
given in Tables 3 and 4.
1 0
:
3~
Table 3
Type of preparation Dose of treatment for Effectiveness of ;~
each caterpillar, in destruction of
microlitres caterpillars,
. in %
1. Anti-hem~cy~ic serum 0.25 30
ditto 0.5 32 ~.
ditto 0.75 40
ditto 1.0 40 :
ditto 1.25 50 ;~
- ditto 1.50 58
ditto 1.75 70
ditto 2.5 98
: ditto 4.5 100 :
2. Control experi- ~:
ment (distilled
water 0.25 0 :
3. Ditto 4.5 0 :.
,
33~
Table 4
No. Type of preparation Dose of txeatment Effectiveness
for each of destruction -~
caterpillar of cater
pillars in %
l. Anti-hemocytic 0.25~1~0.016 ml of a -. .
serum with 0.1% water suspension of ~ ~:
entomopathogenic bacteria 50
bacteria Bac. 0.5~1~0.016 ml of a :~:
: thuringiensis 0.1% water suspension ` ~:
cerëus~~var. of bacteria 67
qalleriae . ~.
1.0~1+0.016 ml of a ` :
- 0.1% water suspension
. of bacteria 83
2.5~1+0.016 ml of a
0.1% water suspension ::
of bacteria lO0
4.5~1+0.016 ml of a
0.1% water suspension
of bacteria ` lO0 ~ :
: 2. Cdntroll.
experiment
(without :~
treatment) - ~`
3. Control ~
experiment ` `
` (distilled ~ .
: water) 2.5 ~l 0 :
` ' " ~
~ :20
'
, ' .
::
~.
-`~ 'I 12 -
. ,, ,~
