Brazzel, J. R.

Davich, T. B.

Raven Klaus

Texas A&M University

MISCELLANEOUS PUBLICATION 353 
TEXAS AGRICULTURAL EXPERIMENT STATION -THE TEXAS A&M COLLEGE SYSTEM 
R. D. Lewis, Director, College Station, Texas, April 30, 1959 
REARING BOLL WEEVILS ON AN ARTIFICIAL DIET 
J. R. Brazzel, T. B. Davich and Klaus Raven* 
-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-
StJMrVl.ARY 
A technique was developed for rearing large numbers of boll weevils from 
egg to adult in lots of 50 on an artificial diet under close confinement. The 
addition of a mixture of sorbic acid and methyl para-hydroxybenzoate (methyl para-
ben) to the diet eliminated the necessity of asceptic procedures. About 70 percent 
of the eggs transferred to the diet developed into adults in 14 to 18 days. Weevils 
reared on this media were uniform in size and averaged about 12 milligrams in 
weight. 
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Introduction 
Boll weevils for use in laboratory toxicity studies were obtained in the 
past by collecting adults from infested cotton fields or by picking infested squares 
from the field and holding them in the laboratory until the weevils emerged. Recent 
work reported by Rainwater & Gaines (1951), Reiser et. al (1953) and other workers 
showed that considerable variation existed among weevils collected from different 
sources and at different times of the year. With field-collected insects, boll 
weevil studies were restricted to the period of the year when the insect was avail-
able in required abundance in the field. 
-The occurrence of resistance to chlorinated hydrocarbon insecticides in 
the boll weevil in Louisiana (Roussel & Clower, 1957) and later in Texas (Walker 
~. a1, 1956) resulted in an intensified research program along biological, ecologi-
cal, physiological and toxicological lines. These studies required a constant 
supply of standardized cultures of the boll weevil. Vanderzant & Davich (1958) re-
pO::"J:,ed a semi-synthetic larval diet and technique for rearing weevils from egg to 
adill-t in individual containers under asceptic conditions. Earle (1959) modified 
this larval diet by substituting acetone-extracted cotton squares for the soybean 
pro~ein and adding a mixture of sorbic acid and methyl paraben to reduce interfer-
ence with growth and development by contaminating microorganisms. With these 
modifications, he was able to rear weevils in individual shell vials under non-
aseptic conditions. Labor costs were excessive for rearing the large number of 
weevils necessary for research work by the procedures cited above. 
The technique for mass rearing weevils on a larval diet in groups of 50 
individuals per rearing container under non-aseptic conditions is reported here. 
*Respectively, associate professor, Department of Entomology, Texas Agricultural 
Experiment Station; entomologist, Entomology Research Division, Agricultural Re~ 
search Service, U. S. Department of Agriculture; and graduate student, Department 
of Entomology. 
-2-
Experimental Procedure 
Several chemicals were tested to determine their effectiveness as anti-
~6~'~~_ agents. The materials included Roccal, sorbic acid, sodium benzoate and 
, ethyl, propyl and butyl parabens. These chemicals were tested as surface-
zing agents by applying them to the surface of the sterilized diet and by 
~m~'~T.ing them into the unsterilized diet. Petri dishes were used as rearing 
I1teL1n.~rs. The effect of the various inhibitors on the boll weevils was tested by 
larvae aseptically in media which contained various concentrations of the 
~."UooIl.g tested. Data were obtained on toxic effects, weevil size and duration 
developmental period. 
Eggs were transferred to the medium in a liquid suspension and by means 
a brush. The liquid transfer of eggs consisted of pipetting approximately 50 
randomly over the smooth surface of the medium in a solution of 3,000 ppm 
• The brush technique involved transfer of the eggs from the same concentra-
of Roccal solution into individual holes made in the diet. The larval feeding 
were spaced at approximately 1 centimeter intervals and were made with a de-
which allowed punChing of all holes in one dish of diet in one operation. 
Eggs were obtained by dissecting cotton squares that had been exposed to 
for 24 hours in an oviposition cage. 
Results 
None of the chemical inhibitors tested by application to the surface of 
medium reduced the growth of microorganisms satisfactorily. The transfer of 
on the surface of the medium in Rocca! solution also was unsatisfactory because 
t~ failure of the larvae to enter the diet and because of excessive cannibalism. 
Dosage mortality lines were obtained for all th~inbibitors incorporated 
the diet except Roccal. Results indicated that less than 5 percent mortality 
d from exposure of the larvae to 0.2 percent sorbic acid and 0.15 percent 
paraben. The other three parabens tested were considerably more toxic. The 
of sodium benzoate to the weevil was lower, but inhibition of microorgan-
was inferior to the other materials. Sorbic acid or methyl paraben used alone 
dosages that would kill 5 percent of the larvae were not effective inhibitors; 
, when used in combination, these tvl0 r.aaterials controlled the growth of 
satisfactorily. 
The combination of sorbic acid at 0.2 percent and methyl paraben at 0.15 
included in the diet caused less than 5 percent weight reduction in the 
and had no measurable effect on the duration of the developmental stages. 
inhibitors did not reduce significantly the percentage weevil eggs which 
• A comparison of these inhibitors in the soybean protein diet (Vanderzant, 
with the cotton square protein diet (Earle, 1959) indicated a higher level of 
ial inhibition when the protein source was acetone-extracted cotton squares. 
results were obtained when acetone-extracted cotton leaves were substituted 
a portion of the soybean protein, except that the weevils weighed approximately 
igram less than in case of individuals reared on the extracted-square diet. 
In addition to incorporation of inhibitors in the diet, several other 
ices were followed to decrease the incidence of contamination. Eggs were 
sterilized with a 3,000 ppo solution of Roccal immediately before transfer 
the diet. Eggs could be transferred directly from the Roccal solution to the 
• All petri plates were sterilized in an oven for at least 1 hour at 1800 c. 
-3-
~th of the diet in the petri plates and the moisture content were critical 
in reducing the growth of microorganisms. The diet was poured to a depth 
6 millimeters in the petri plates. This depth was sufficient to prevent 
larvae from breaking out of the feeding cell and wandering about over the sur-
ot the diet and allowed the diet to dry sufficiently to arrest local spots of 
~~'W.~'U' The drying of the diet was done by covering dishes immediately after 
'_'W.""U with cardboard dessicator tops. After 24 hours these tops were replaced 
the glass petri plate cover. 
Most of the weevils emerged in 14 to 18 days after the eggs were placed 
diet • With the media poured to a depth of 6 millimeters, the adults were 
to emerge from the pupal cell unassisted. Adults were obtained from about 70 
of' the eggs implanted in the diet. 
Detailed instructions for mixing and the ingredients necessary for ade-
boll weevil larval diet to pour 12 petri plates follow: 
1. Weigh the following ingredients into container 1: 20.0 gm of square 
or leaf meal and 25.0 gm of soybean protein. 
2. Weigh the following ingredients into container 2: 25.0 gm sucrcse, 
SIll L cysteine HCL, 0.5 gm Glycine, 0.25 gm Cholesterol, 5.0 gIn Wesson Salts, 
am Sodium Algenate; and 5.0 gIn Brewers yeast. 
3. Weigh 15.0 gm agar into container 3. 
4. Heat 430 ml of distilled H20 to boiling. VIhile the vlater is heating, 
following liquid ingredients are measured into the mixing bowl of a 1 quart 
blender: 10.0 ml of inhibitor solution. (The inhibitor solution is prepared 
20.0 gm of sorbic acid and 15.0 gm of methyl paraben in 170.0 ml of 
j 14.0 ml of KOH (10 percent); 5.0 ml of vitamin-aolution (constituents 
by Vanderzant, et. al , 1957); and 2.5 ml of Mazola oil. 
5. After the water begins to boil, add the agar in cont.a iner 3 and stir 
dirsolv€·d., Pour the dissolved agar solution into the mixej.' p-,nd mix at low 
8!; ",~e i ngredients in container 2 are added slo'\vly. After this G'Jlution is 
tro:::"')'Jgbly, increase the mixer speed and add the content of container 1 slow-
~nlen the contents have been mixed thoroughly, pour the media into sterilized 
plates. This amount of media is sufficient to pour 12 plates. 
After the media has solidified, holes are made in the top for the eggs. 
are s'~erilized in 3,000 ppm solution of Roccal and placed individually in the 
~oles in the media with a camelis hair brush. No further aseptic precau-
are necessary during the transfer of eggs. The rearing containers were 
with a dessicator top of cardboard for 24 hours and replaced 'vith the glass 
-4-
Literature Cited 
Earle, N. W.,_ R. C. Gaines and J. S. Roussel.. 1959. A larval diet for 
boll weevil containing an acetone powder of cotton squares. Jour. Econ. Ent. 
a). 
Rainwater, C. F. and J. C. Gaines. 1951.. Seasonal. decline in the effec-
of certain insecticides against the boll weevil. Jour. Econ. Ent. 
:974-16. 
Reiser, Raymond, Danville S. Chadburne, Kenneth A. Kuiken, C. F. Rain-
and E. E. Ivy. 1953. Variations in the lipid content of the boll weevil and 
variation in its resistance to insecticides. Jour. Econ. Ent. 
:337-40. 
Vanderzant, Erma S., Dunndappa Kerur and Raymond Reiser. 1957. The role 
..... _. tatty acids in the development of the pink bollworm. Jour. Econ. Ent. 
Vanderzant, Erma S. and T. B. Davich. 1958. Laboratory rearing of the 
weevU. A sa.tisfactory larvaJ. diet and oviposition studies. Jour. Econ. Ent. 
:288-91. 
Walker, J. K. Jr., B. G. Hightower, R. L. Hanna and D. F. Martin. 1956. 
of boll weevils resistant to chlorinated hydroca.rbons. Texas Agric. Expt. 
Prog. Rpt. 1902. 
- 0 -


English

Rearing Boll Weevils on an Artificial Diet.

Texas A&amp;M Repository

MISCELLANEOUS PUBLICATION 353 TEXAS AGRICULTURAL EXPERIMENT STATION -THE TEXAS A&M COLLEGE SYSTEM R. D. Lewis, Director, College Station, Texas, April 30, 1959 REARING BOLL WEEVILS ON AN ARTIFICIAL DIET J. R. Brazzel, T. B. Davich and Klaus Raven* -00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-StJMrVl.ARY A technique was developed for rearing large numbers of boll weevils from egg to adult in lots of 50 on an artificial diet under close confinement. The addition of a mixture of sorbic acid and methyl para-hydroxybenzoate (methyl para-ben) to the diet eliminated the necessity of asceptic procedures. About 70 percent of the eggs transferred to the diet developed into adults in 14 to 18 days. Weevils reared on this media were uniform in size and averaged about 12 milligrams in weight. -00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-Introduction Boll weevils for use in laboratory toxicity studies were obtained in the past by collecting adults from infested cotton fields or by picking infested squares from the field and holding them in the laboratory until the weevils emerged. Recent work reported by Rainwater & Gaines (1951), Reiser et. al (1953) and other workers showed that considerable variation existed among weevils collected from different sources and at different times of the year. With field-collected insects, boll weevil studies were restricted to the period of the year when the insect was avail-able in required abundance in the field. -The occurrence of resistance to chlorinated hydrocarbon insecticides in the boll weevil in Louisiana (Roussel & Clower, 1957) and later in Texas (Walker ~. a1, 1956) resulted in an intensified research program along biological, ecologi-cal, physiological and toxicological lines. These studies required a constant supply of standardized cultures of the boll weevil. Vanderzant & Davich (1958) re-pO::"J:,ed a semi-synthetic larval diet and technique for rearing weevils from egg to adill-t in individual containers under asceptic conditions. Earle (1959) modified this larval diet by substituting acetone-extracted cotton squares for the soybean pro~ein and adding a mixture of sorbic acid and methyl paraben to reduce interfer-ence with growth and development by contaminating microorganisms. With these modifications, he was able to rear weevils in individual shell vials under non-aseptic conditions. Labor costs were excessive for rearing the large number of weevils necessary for research work by the procedures cited above. The technique for mass rearing weevils on a larval diet in groups of 50 individuals per rearing container under non-aseptic conditions is reported here. *Respectively, associate professor, Department of Entomology, Texas Agricultural Experiment Station; entomologist, Entomology Research Division, Agricultural Re~ search Service, U. S. Department of Agriculture; and graduate student, Department of Entomology. -2-Experimental Procedure Several chemicals were tested to determine their effectiveness as anti-~6~'~~_ agents. The materials included Roccal, sorbic acid, sodium benzoate and , ethyl, propyl and butyl parabens. These chemicals were tested as surface-zing agents by applying them to the surface of the sterilized diet and by ~m~'~T.ing them into the unsterilized diet. Petri dishes were used as rearing I1teL1n.~rs. The effect of the various inhibitors on the boll weevils was tested by larvae aseptically in media which contained various concentrations of the ~."UooIl.g tested. Data were obtained on toxic effects, weevil size and duration developmental period. Eggs were transferred to the medium in a liquid suspension and by means a brush. The liquid transfer of eggs consisted of pipetting approximately 50 randomly over the smooth surface of the medium in a solution of 3,000 ppm • The brush technique involved transfer of the eggs from the same concentra-of Roccal solution into individual holes made in the diet. The larval feeding were spaced at approximately 1 centimeter intervals and were made with a de-which allowed punChing of all holes in one dish of diet in one operation. Eggs were obtained by dissecting cotton squares that had been exposed to for 24 hours in an oviposition cage. Results None of the chemical inhibitors tested by application to the surface of medium reduced the growth of microorganisms satisfactorily. The transfer of on the surface of the medium in Rocca! solution also was unsatisfactory because t~ failure of the larvae to enter the diet and because of excessive cannibalism. Dosage mortality lines were obtained for all th~inbibitors incorporated the diet except Roccal. Results indicated that less than 5 percent mortality d from exposure of the larvae to 0.2 percent sorbic acid and 0.15 percent paraben. The other three parabens tested were considerably more toxic. The of sodium benzoate to the weevil was lower, but inhibition of microorgan-was inferior to the other materials. Sorbic acid or methyl paraben used alone dosages that would kill 5 percent of the larvae were not effective inhibitors; , when used in combination, these tvl0 r.aaterials controlled the growth of satisfactorily. The combination of sorbic acid at 0.2 percent and methyl paraben at 0.15 included in the diet caused less than 5 percent weight reduction in the and had no measurable effect on the duration of the developmental stages. inhibitors did not reduce significantly the percentage weevil eggs which • A comparison of these inhibitors in the soybean protein diet (Vanderzant, with the cotton square protein diet (Earle, 1959) indicated a higher level of ial inhibition when the protein source was acetone-extracted cotton squares. results were obtained when acetone-extracted cotton leaves were substituted a portion of the soybean protein, except that the weevils weighed approximately igram less than in case of individuals reared on the extracted-square diet. In addition to incorporation of inhibitors in the diet, several other ices were followed to decrease the incidence of contamination. Eggs were sterilized with a 3,000 ppo solution of Roccal immediately before transfer the diet. Eggs could be transferred directly from the Roccal solution to the • All petri plates were sterilized in an oven for at least 1 hour at 1800 c. -3-~th of the diet in the petri plates and the moisture content were critical in reducing the growth of microorganisms. The diet was poured to a depth 6 millimeters in the petri plates. This depth was sufficient to prevent larvae from breaking out of the feeding cell and wandering about over the sur-ot the diet and allowed the diet to dry sufficiently to arrest local spots of ~~'W.~'U' The drying of the diet was done by covering dishes immediately after '_'W.""U with cardboard dessicator tops. After 24 hours these tops were replaced the glass petri plate cover. Most of the weevils emerged in 14 to 18 days after the eggs were placed diet • With the media poured to a depth of 6 millimeters, the adults were to emerge from the pupal cell unassisted. Adults were obtained from about 70 of' the eggs implanted in the diet. Detailed instructions for mixing and the ingredients necessary for ade-boll weevil larval diet to pour 12 petri plates follow: 1. Weigh the following ingredients into container 1: 20.0 gm of square or leaf meal and 25.0 gm of soybean protein. 2. Weigh the following ingredients into container 2: 25.0 gm sucrcse, SIll L cysteine HCL, 0.5 gm Glycine, 0.25 gm Cholesterol, 5.0 gIn Wesson Salts, am Sodium Algenate; and 5.0 gIn Brewers yeast. 3. Weigh 15.0 gm agar into container 3. 4. Heat 430 ml of distilled H20 to boiling. VIhile the vlater is heating, following liquid ingredients are measured into the mixing bowl of a 1 quart blender: 10.0 ml of inhibitor solution. (The inhibitor solution is prepared 20.0 gm of sorbic acid and 15.0 gm of methyl paraben in 170.0 ml of j 14.0 ml of KOH (10 percent); 5.0 ml of vitamin-aolution (constituents by Vanderzant, et. al , 1957); and 2.5 ml of Mazola oil. 5. After the water begins to boil, add the agar in cont.a iner 3 and stir dirsolv€·d., Pour the dissolved agar solution into the mixej.' p-,nd mix at low 8!; ",~e i ngredients in container 2 are added slo'\vly. After this G'Jlution is tro:::"')'Jgbly, increase the mixer speed and add the content of container 1 slow-~nlen the contents have been mixed thoroughly, pour the media into sterilized plates. This amount of media is sufficient to pour 12 plates. After the media has solidified, holes are made in the top for the eggs. are s'~erilized in 3,000 ppm solution of Roccal and placed individually in the ~oles in the media with a camelis hair brush. No further aseptic precau-are necessary during the transfer of eggs. The rearing containers were with a dessicator top of cardboard for 24 hours and replaced 'vith the glass -4-Literature Cited Earle, N. W.,_ R. C. Gaines and J. S. Roussel.. 1959. A larval diet for boll weevil containing an acetone powder of cotton squares. Jour. Econ. Ent. a). Rainwater, C. F. and J. C. Gaines. 1951.. Seasonal. decline in the effec-of certain insecticides against the boll weevil. Jour. Econ. Ent. :974-16. Reiser, Raymond, Danville S. Chadburne, Kenneth A. Kuiken, C. F. Rain-and E. E. Ivy. 1953. Variations in the lipid content of the boll weevil and variation in its resistance to insecticides. Jour. Econ. Ent. :337-40. Vanderzant, Erma S., Dunndappa Kerur and Raymond Reiser. 1957. The role ..... _. tatty acids in the development of the pink bollworm. Jour. Econ. Ent. Vanderzant, Erma S. and T. B. Davich. 1958. Laboratory rearing of the weevU. A sa.tisfactory larvaJ. diet and oviposition studies. Jour. Econ. Ent. :288-91. Walker, J. K. Jr., B. G. Hightower, R. L. Hanna and D. F. Martin. 1956. of boll weevils resistant to chlorinated hydroca.rbons. Texas Agric. Expt. Prog. Rpt. 1902. - 0 -

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Rearing Boll Weevils on an Artificial Diet.

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Rearing Boll Weevils on an Artificial Diet.

Abstract

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Full text

Available Versions

Texas A&M University

Texas A&amp;M Repository

Texas A&M Repository