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Inhibition of DNA synthesis in boll weevils (Anthonomus grandis boheman) sterilized by dimilin
PASTICIDE
BIOCHEMISTRY
Inhibition
4ND
PHYSTOLOGY
7, i%-;i6.3
11977)
of DNA Synthesis in Boll Weevils (Ant~onomus Boheman) Sterilized by Dimilin NORMAN
MITLIN,
Boll
ResearchLaboratory,
Weevil
GLENN
Mississippi
WIY~UL, Agricultural State,
Mississippi
grandis
AND JACK W. HAYNES Research 39762
Service,
USDA,
Received April 26, 1976; accepted January 14, 1977 When boll weevils, Anthonomus grandis Boheman, were treated with dimilin (N- (4-chlorophenyl)-N’-(2,6-difluorobenzoyl)urea), the biosynthesis of deoxyribonucleic acid was inhibited in the female, but neither ribonucleic acid nor protein synthesis was affected. Treated males showed a differencein effect in lipoprotein synthesis, whereas no significant difference wae demonstrated with females. Testicular growth was inhibited in some of the males. Diminishment of sexual function may therefore result in part from inhibit,inn of hiosynt,heais nf DNA by climilin. INTllODUCTION
It was reported earlier (1) that dimilin (N-(4-chlorophenyl)-N’-(2,6-difluorobenzoapparently sterilized the boll ylhma), weevil. The authors surmised that the sterilization was caused by the transfer of dimilin from the treated male to the female. In the course of further evaluating the effect of dimilin as a chemosterilant of the boll weevil, Anthonomus grandis Roheman, we noted the juvenilization of the gonads typical of sterilization in a number of insects. In a number of instances whenever there was no hatch of eggs, the growth of the gonads of the treated male parent had been inhibited with accompanying destruction of the germaria. Although dimilin is known to affect chitin synthesis (24), the growth regulator might also inhibit nucleic acids and protein synthesis since the alkylating agents and antimetabolites that make up the bulk of known chemosterilants are such inhibitors (5, 6). A study was undertaken to determine
whether the chemical was indeed an inhibitor. We used 14C-labeled thymidine and uridine as precursors of DNA and RNA, respectively, and followed their incorporation into the nucleic acids. Although uridine does not label RNA only, it is effective because RNA is synthesized rapidly after adult eclosion; DNA is synthesized relatively slowly (7). To follow protein synthesis, we used p%]lysine. MATERIALS
AND
METHODS
The radiotracers [2J4C]uridine, [2-14C]thymidine, and [UJ4C]lysine were obtained commercially. The radiopurity of the uridine and thymidine was checked by paper chromatography in (i) ethyl acetate-formic acid-water (65 : 5 : 35), (ii) in butanol-acetic acid-water (80 : 12 : 30), (iii) in butanol-0.6 N ammonia (6:1), and (iv) in water. The purity of the compounds was found to be greater than 99%. The purity of the leucine was checked by paper chromatography in N-butanol-pyridineacetic acid-water (75 : 50 : 15 : 55) and phe559
Copyright rights
All
@ 1977 by Academic of reproduction in my
Prese, Inc. form reserved.
ISSN
0048-3575
fQ/?ecl
of
Ct.470 TM-60.$0
on
Nf,WlJl
hel~yed
Boll Weevils (3 replications) replications)”
when Males were Dipped or Females were Dipped
Trentnlentb
Cunndetive (70) mortality of treated inserts at day -__I
Tc? x 1-o uflxx-0 1‘0 Xlic? 113 x 11 0
7
14
21
IO 11 lfi 14
32 32 2k 18
36 37 “6 21
*I Dips were 1-2 hr apart,. b T , trPRtCd~ , u ? untrmtrd.
Squnres
~ttldt
5-o’ Tknes 3 Times C/i
rercrntngc egg hatch nt day II_ 7 14 21
I’ercrntage adult rmrrgenw at day --.~--7 14 21
0 85 0 80 .---
0 30 0 RR
a3 81 4 87
were
no 71 15 80
fed post-trentmcnt.
32 3.5 0 31
41 40 II 35
had obsrrved in pr~~linlini~ry Lcsts with fewer dips that the testes might be affected and to ensure adequate exposure to the chemical we increased the number of dips. After the last dip, the weevils were caged individually with untreated weevils of the opposite sex, fed squares (flower buds), and held at 30°C. At 7, 14, and 21 days post-treatment, mortalities were recorded, and samples of 50 eggs/week were collected for determination of egg hat,ch and of adult emergence. Biochemical Procedures
When t,he bioc~hrmic:~I tcstx wcrc to 1)c the weevils were held for 2 1~1. aft#er nol-water (100:39, w/v) and found to be t,he final dip, then when t#hey had rrcovereti :tt least 99y0 purr. The radiochemicals inje:ct,rd wit,h one ot’t,hr radiot racers. Insect,s were used without, further purification. injected witfh lahelecl uridinc were held f01 For injection, both t,hymidine and uridine 20 min ant1 then cluiakly frozen. Those were dissolved in water; lysine was disinj&ed wit,h the %Llabele(l t.hymidinr solved in 0.01 N HCl. Dimilin (provided were held for 4s hr before freezing, ant1 1)~ the Thompson-Hayward Chemical Co., Kansas City, Missouri) was dissolved in those injected with l”
DIRIILIN
FIG.
INHIBITION
OF DNA
1. Exurnplp and
BIOSYNTHESIS
qf grow&inhibited of normal male
was treated with 1.0 N KOH and held for 3 days at 37°C (12). After the RNA was removed the DNA was hydrolyzed with formic acid to free the constituent bases. The tissue hydrolysate was chromatographed on Whatman No. 1 filter paper and the chromatograms were developed in isopropanol-2 N HCl. After locating the spots under a shortwave uv lamp, the spots were cut out and each was placed in scintillation fluid and counted. Four replications of each test were run and the results were analyzed by analysis of variance and significance determined by Duncan’s multiple-range test (13). Because preliminary tests showed evidence of some incorporation of 14C-labeled thymidine in the residual protein fraction, something of an anomaly since thymidine is presumably a specific precursor of DNA, we hydrolyzed the protein fraction with 6 N NC1 in sealed evacuated tubes that were heated for 18-20 hr at 110°C. After hydrolysis, the residual humin was filtered off, and the hydrolyzed protein solution
IN
testes o.ftreated weroils irigh,t).
BOLL
WEEVILS
mnlrs
56 I
clef/)
was taken to dryness, washed twice with distilled water, taken up in ca. 2 ml of water, and prepared for gas-liquid chromatography by the method of Zumwalt el al. (14) in which amino acids are converted to N-trifluoroacetyl (N-TFA) n.-butyl ester derivatives. To determine whether the protein amino acids were labeled, we chromatographed them on a Micro-Tee Model GC-2000R dual hydrogen flame gas chromatograph with a stream splitter. Each amino acid was captured on cartridges and processed as described by Watson and Williams (16). RESULTS
AND
DISCUSSION
Results of Treatment
The results of the treatment are shown in Table 1. Dimilin, as in the test of Moore and Taft (l), was more effective in the female weevils. Egg hatch was reduced as a result of treatment of either sexes, but the treated males showed an almost complete recovery by the end of the third week. The females were more affected; egg
\II’I‘I,lN.
.5(i’J
\VlYGUId,
ANI)
TABLE l&~lios
oJ Incurporaliorb
rrAYuF:s
2
Hadiolabeled Precursor into the Indicated I’raclions were Treated with Dimilin or Untreateda
aJ’trr 11~1,Boll
Weevils
Precursor
Fractjion
-
Acid-soluble
___-. Treated
Males Untreated
-~. Treated
_.
Untreated
Nucleic acid
2.11 zk 0.21”
0.96 f 0.26’)
1.82 f 0.16”
[W]Uridine
Acid-soluble Nucleic acid
522 f 0.47a
4.32 f 0.72*
4.66 f 0.303 4.04 f 0.74z
[W]Lysine
Free amino acid I,ipoprot,ein
4.78 f 0.4@
3.91 f 0.2”
1.56 f 0.19c 1.82 f 0.220
Free amino acid _~___.~--._ Protein
2.0.; f 0.69~ 1.70 f 0.68;
WW’hymidine
-
_
I_~_~_
-. --._- _
0.21 f o.or,r
--... .-... -----.-.. ___.. -.
~-
Females
1.19 f 0.20”
0.19 f 0.01”
.- - ..~~_~_.
0 Ratios followed by ditl’erent, sllperscripl, let,ters are different, at the 5’% level of aignifirance fcjr the indicated radiotracer.
hatch remained low for 3 weeks after treatment. Percentage emergence of adult progeny followed the pattern of egg hatch. Moore and Taft (1) suggested that, male weevils treated with dimilin transferred the chemical to females by cont,act and ascribed the resultant low egg hatch and emergence to the action of the mat,erial on the female gonads, not on the male reproductive organs. However, we found that successive dipping of males in a solution of dimilin caused a 30% reduction of the size of the testes of 50% of the treated males and a 50% reduction in the size of 5% of the males at 2 weeks posttreatment (Fig. 1). Also, in normal insects, the testes were opaque only in the region of the germarian ; in treated males the testes were opaque throughout. Thus, dimilin does affect the reproductive capacity of male boll weevils directly, albeit to a much lesser extent, and is therefore a true sterilant. Incorporation Studies
There were no significant differences in the incorporation of uridine into any of the fractions from treated or untreated insect,s (Table 2). Neither were there any
differences between sexes. This result, is similar t,o those obtained in :m earlier study with the alkylating agent busulfan (6). Also, it is generally similar to results obtained with other organisms (17). RNA synthesis is only slightly affectted by nlkylating agents. Treatment with dimilin did significantl? (P = 0.05) slow the incorporation of labeled thymidine int#o the nucleic acid fraction of weevils of both sexes, which indicates that the growth regulator inhibits synthesis of that nucleic acid because thymidine is specifically a precursor of DNA. As the ratios indicated, the amount of radioactivity in the acid-soluble fraction from treated insects increased, indicating there was some interference with the utilization of the precursors of polynucleotides. Similar effects were previously noted with busulfan (6), but busulfan caused greater inhibition than dimilin. Measurement of the percentage of radioactivity incorporated into the individual bases of the DNAs showed that about 90% of the recovered radioactivity was incorporated into thymine. The other 10% was divided almost equally among the three other bases. This was probably an
DIMILIN
INHIBITION
OF
DNA
BIOSYNTHESIS
artifact given the tendency of radiation to spread out along a paper chromatogram. There was no significant difference (P = 0.05) among the treatment or methods, except in the instance of females treated with dimilin which showed significantly less incorporation. This appears to be in accord with the results reported above. A significant difference was not demonstrated in dimilin-treated males, probably because of the relatively large statistical error. We could detect no radioactivity in any of the individual protein amino acids in the tests with thymidine despite the fact that the protein fraction, in its entirety, was consistently labeled, though at a relatively low level. Possibly the incorporation shown was real but below the limits of detectability in the individual amino acids. Possibly such incorporation may occur as a result of thymidine catabolism. Dimilin caused no significant decrease in incorporation of C’4C]lysine into the protein fraction of either sex. However, there was a significant difference in incorporation between treated and untreated weevils in the lipoprotein fraction of the male. This is something of an anomaly since we have shown earlier that the incorporation of labeled lysine appeared to precede its incorporation into protein (15), and one would expect differences occurring in one fraction to be repeated in the other. Since, however, the relative standard errors were higher in the protein fractions, they may have served to mask such a difference. ACKNOWLEDGMENTS
We wish to thank Gertrude Gibson and Betty Nail both of this laboratory for their help in oonducting the tests. REFERENCES
1. R. F. Moore, Jr. and H. M. Taft, Boll Weevils: Chemosterilization of both sexes with bu&fan plus Thompson-Hayward TH-6040, J. Econ. Entomol. 68, 96 (1975).
IN
BOLL
WEEVILS
563
2. R. Mulder and M. J. Gijswijt, The laboratory evaluation of two promising insecticides which interfere with cuticle deposition. Pestic. Sci. 4, 737 (1973). 3. L. C. Post and W. R. Vincent, A new insecticide inhibits chitin synthesis, Natllrwissenschaften 60, 431 (1973). 4. I. Ishaaye and J. E. C&da, Dietary TH-6040 alters composition and enzyme activity of housefly larvae cuticle, Pestic. Biochem. Physiol.
4, 484 (1974).
5. N. Mitlin, The physiology and toxicology of chemosterilants, in “Proceedings of the XIIth International Congress on Entomology,” London, 1964 (1965). 6. N. Mitlin and G. Wiygul, Synthesis of nucleic acid and protein in the boll weevil fed with busulfan, Bnn. Entomol. Sot. Amer. 64, 822 (1971). 7. N. Mitlin and G. Wiygul, Incorporation and metabolism of ‘%-labeled tryptophan-3 in the boll weevil Anthonomus grand& Boheman, Comp. Biochem.. Physiol. 30, 375 (1969). 8. A. C. Bartlett, Genetic markers in the boll weevil, J. Hered. 58, 159 (1967). 9. R. T. Cast, Oviposition and fecundity of boll weevils in mass-rearing cultures. J. Econ. Entomol.
59, 173 (1966).
10. W. C. Schneider, Phosphorus compounds in animal tissues. III. A comparison of methods for the estimation of nucleic acids, J. Bid. Chem. 164, 747 (1946). 11. I>. T. Mahin and R. T. Lofberg, A simplified met,hod of sample preparation for determination of tritium, carbon-14, or sulfur-35 in blood or t,issue by liquid scint,illation counting, Anal. Biochem. 16, 500 (1966). 12. G. Schmidt and S. J. Thannhauser, A method for the det,ermination of desoxyribonucleic acid, ribonucleic acid and phospho-protein in animal tissues, J. Biol. Chem. 161, 83 (1945). 13. D. B. Duncan, Multiple range and multiple F tests, Biometrics 11, 1 (1955). 14. R. W. Zumwalt, D. Roach, and C. W. Gehrke, Gas-liquid chromatography of amino acids in biological substances, J. Chromabgr. 53, 171 (1970). 1.5. N. Mitlin, G. Wiygul, and G. L. Lusk, Incorporation of lysine-6-04 int’o the protein of the adult boll weevil, Anthonomus grads, J. Insect Physiol. 14, 1277 (1968). 16. G. R. Watson and J. P. Williams, An improved method for collection and measurement of radioactivity in compounds separated by gasliquid chromatography, J. Chromatogr. 67, 221 (1972). 17, G. P. Wheeler, Some biochemical effects of dkylating agents, Fed. Proc. 26, 885 (1967).
BIOCHEMISTRY
Inhibition
4ND
PHYSTOLOGY
7, i%-;i6.3
11977)
of DNA Synthesis in Boll Weevils (Ant~onomus Boheman) Sterilized by Dimilin NORMAN
MITLIN,
Boll
ResearchLaboratory,
Weevil
GLENN
Mississippi
WIY~UL, Agricultural State,
Mississippi
grandis
AND JACK W. HAYNES Research 39762
Service,
USDA,
Received April 26, 1976; accepted January 14, 1977 When boll weevils, Anthonomus grandis Boheman, were treated with dimilin (N- (4-chlorophenyl)-N’-(2,6-difluorobenzoyl)urea), the biosynthesis of deoxyribonucleic acid was inhibited in the female, but neither ribonucleic acid nor protein synthesis was affected. Treated males showed a differencein effect in lipoprotein synthesis, whereas no significant difference wae demonstrated with females. Testicular growth was inhibited in some of the males. Diminishment of sexual function may therefore result in part from inhibit,inn of hiosynt,heais nf DNA by climilin. INTllODUCTION
It was reported earlier (1) that dimilin (N-(4-chlorophenyl)-N’-(2,6-difluorobenzoapparently sterilized the boll ylhma), weevil. The authors surmised that the sterilization was caused by the transfer of dimilin from the treated male to the female. In the course of further evaluating the effect of dimilin as a chemosterilant of the boll weevil, Anthonomus grandis Roheman, we noted the juvenilization of the gonads typical of sterilization in a number of insects. In a number of instances whenever there was no hatch of eggs, the growth of the gonads of the treated male parent had been inhibited with accompanying destruction of the germaria. Although dimilin is known to affect chitin synthesis (24), the growth regulator might also inhibit nucleic acids and protein synthesis since the alkylating agents and antimetabolites that make up the bulk of known chemosterilants are such inhibitors (5, 6). A study was undertaken to determine
whether the chemical was indeed an inhibitor. We used 14C-labeled thymidine and uridine as precursors of DNA and RNA, respectively, and followed their incorporation into the nucleic acids. Although uridine does not label RNA only, it is effective because RNA is synthesized rapidly after adult eclosion; DNA is synthesized relatively slowly (7). To follow protein synthesis, we used p%]lysine. MATERIALS
AND
METHODS
The radiotracers [2J4C]uridine, [2-14C]thymidine, and [UJ4C]lysine were obtained commercially. The radiopurity of the uridine and thymidine was checked by paper chromatography in (i) ethyl acetate-formic acid-water (65 : 5 : 35), (ii) in butanol-acetic acid-water (80 : 12 : 30), (iii) in butanol-0.6 N ammonia (6:1), and (iv) in water. The purity of the compounds was found to be greater than 99%. The purity of the leucine was checked by paper chromatography in N-butanol-pyridineacetic acid-water (75 : 50 : 15 : 55) and phe559
Copyright rights
All
@ 1977 by Academic of reproduction in my
Prese, Inc. form reserved.
ISSN
0048-3575
fQ/?ecl
of
Ct.470 TM-60.$0
on
Nf,WlJl
hel~yed
Boll Weevils (3 replications) replications)”
when Males were Dipped or Females were Dipped
Trentnlentb
Cunndetive (70) mortality of treated inserts at day -__I
Tc? x 1-o uflxx-0 1‘0 Xlic? 113 x 11 0
7
14
21
IO 11 lfi 14
32 32 2k 18
36 37 “6 21
*I Dips were 1-2 hr apart,. b T , trPRtCd~ , u ? untrmtrd.
Squnres
~ttldt
5-o’ Tknes 3 Times C/i
rercrntngc egg hatch nt day II_ 7 14 21
I’ercrntage adult rmrrgenw at day --.~--7 14 21
0 85 0 80 .---
0 30 0 RR
a3 81 4 87
were
no 71 15 80
fed post-trentmcnt.
32 3.5 0 31
41 40 II 35
had obsrrved in pr~~linlini~ry Lcsts with fewer dips that the testes might be affected and to ensure adequate exposure to the chemical we increased the number of dips. After the last dip, the weevils were caged individually with untreated weevils of the opposite sex, fed squares (flower buds), and held at 30°C. At 7, 14, and 21 days post-treatment, mortalities were recorded, and samples of 50 eggs/week were collected for determination of egg hat,ch and of adult emergence. Biochemical Procedures
When t,he bioc~hrmic:~I tcstx wcrc to 1)c the weevils were held for 2 1~1. aft#er nol-water (100:39, w/v) and found to be t,he final dip, then when t#hey had rrcovereti :tt least 99y0 purr. The radiochemicals inje:ct,rd wit,h one ot’t,hr radiot racers. Insect,s were used without, further purification. injected witfh lahelecl uridinc were held f01 For injection, both t,hymidine and uridine 20 min ant1 then cluiakly frozen. Those were dissolved in water; lysine was disinj&ed wit,h the %Llabele(l t.hymidinr solved in 0.01 N HCl. Dimilin (provided were held for 4s hr before freezing, ant1 1)~ the Thompson-Hayward Chemical Co., Kansas City, Missouri) was dissolved in those injected with l”
DIRIILIN
FIG.
INHIBITION
OF DNA
1. Exurnplp and
BIOSYNTHESIS
qf grow&inhibited of normal male
was treated with 1.0 N KOH and held for 3 days at 37°C (12). After the RNA was removed the DNA was hydrolyzed with formic acid to free the constituent bases. The tissue hydrolysate was chromatographed on Whatman No. 1 filter paper and the chromatograms were developed in isopropanol-2 N HCl. After locating the spots under a shortwave uv lamp, the spots were cut out and each was placed in scintillation fluid and counted. Four replications of each test were run and the results were analyzed by analysis of variance and significance determined by Duncan’s multiple-range test (13). Because preliminary tests showed evidence of some incorporation of 14C-labeled thymidine in the residual protein fraction, something of an anomaly since thymidine is presumably a specific precursor of DNA, we hydrolyzed the protein fraction with 6 N NC1 in sealed evacuated tubes that were heated for 18-20 hr at 110°C. After hydrolysis, the residual humin was filtered off, and the hydrolyzed protein solution
IN
testes o.ftreated weroils irigh,t).
BOLL
WEEVILS
mnlrs
56 I
clef/)
was taken to dryness, washed twice with distilled water, taken up in ca. 2 ml of water, and prepared for gas-liquid chromatography by the method of Zumwalt el al. (14) in which amino acids are converted to N-trifluoroacetyl (N-TFA) n.-butyl ester derivatives. To determine whether the protein amino acids were labeled, we chromatographed them on a Micro-Tee Model GC-2000R dual hydrogen flame gas chromatograph with a stream splitter. Each amino acid was captured on cartridges and processed as described by Watson and Williams (16). RESULTS
AND
DISCUSSION
Results of Treatment
The results of the treatment are shown in Table 1. Dimilin, as in the test of Moore and Taft (l), was more effective in the female weevils. Egg hatch was reduced as a result of treatment of either sexes, but the treated males showed an almost complete recovery by the end of the third week. The females were more affected; egg
\II’I‘I,lN.
.5(i’J
\VlYGUId,
ANI)
TABLE l&~lios
oJ Incurporaliorb
rrAYuF:s
2
Hadiolabeled Precursor into the Indicated I’raclions were Treated with Dimilin or Untreateda
aJ’trr 11~1,Boll
Weevils
Precursor
Fractjion
-
Acid-soluble
___-. Treated
Males Untreated
-~. Treated
_.
Untreated
Nucleic acid
2.11 zk 0.21”
0.96 f 0.26’)
1.82 f 0.16”
[W]Uridine
Acid-soluble Nucleic acid
522 f 0.47a
4.32 f 0.72*
4.66 f 0.303 4.04 f 0.74z
[W]Lysine
Free amino acid I,ipoprot,ein
4.78 f 0.4@
3.91 f 0.2”
1.56 f 0.19c 1.82 f 0.220
Free amino acid _~___.~--._ Protein
2.0.; f 0.69~ 1.70 f 0.68;
WW’hymidine
-
_
I_~_~_
-. --._- _
0.21 f o.or,r
--... .-... -----.-.. ___.. -.
~-
Females
1.19 f 0.20”
0.19 f 0.01”
.- - ..~~_~_.
0 Ratios followed by ditl’erent, sllperscripl, let,ters are different, at the 5’% level of aignifirance fcjr the indicated radiotracer.
hatch remained low for 3 weeks after treatment. Percentage emergence of adult progeny followed the pattern of egg hatch. Moore and Taft (1) suggested that, male weevils treated with dimilin transferred the chemical to females by cont,act and ascribed the resultant low egg hatch and emergence to the action of the mat,erial on the female gonads, not on the male reproductive organs. However, we found that successive dipping of males in a solution of dimilin caused a 30% reduction of the size of the testes of 50% of the treated males and a 50% reduction in the size of 5% of the males at 2 weeks posttreatment (Fig. 1). Also, in normal insects, the testes were opaque only in the region of the germarian ; in treated males the testes were opaque throughout. Thus, dimilin does affect the reproductive capacity of male boll weevils directly, albeit to a much lesser extent, and is therefore a true sterilant. Incorporation Studies
There were no significant differences in the incorporation of uridine into any of the fractions from treated or untreated insect,s (Table 2). Neither were there any
differences between sexes. This result, is similar t,o those obtained in :m earlier study with the alkylating agent busulfan (6). Also, it is generally similar to results obtained with other organisms (17). RNA synthesis is only slightly affectted by nlkylating agents. Treatment with dimilin did significantl? (P = 0.05) slow the incorporation of labeled thymidine int#o the nucleic acid fraction of weevils of both sexes, which indicates that the growth regulator inhibits synthesis of that nucleic acid because thymidine is specifically a precursor of DNA. As the ratios indicated, the amount of radioactivity in the acid-soluble fraction from treated insects increased, indicating there was some interference with the utilization of the precursors of polynucleotides. Similar effects were previously noted with busulfan (6), but busulfan caused greater inhibition than dimilin. Measurement of the percentage of radioactivity incorporated into the individual bases of the DNAs showed that about 90% of the recovered radioactivity was incorporated into thymine. The other 10% was divided almost equally among the three other bases. This was probably an
DIMILIN
INHIBITION
OF
DNA
BIOSYNTHESIS
artifact given the tendency of radiation to spread out along a paper chromatogram. There was no significant difference (P = 0.05) among the treatment or methods, except in the instance of females treated with dimilin which showed significantly less incorporation. This appears to be in accord with the results reported above. A significant difference was not demonstrated in dimilin-treated males, probably because of the relatively large statistical error. We could detect no radioactivity in any of the individual protein amino acids in the tests with thymidine despite the fact that the protein fraction, in its entirety, was consistently labeled, though at a relatively low level. Possibly the incorporation shown was real but below the limits of detectability in the individual amino acids. Possibly such incorporation may occur as a result of thymidine catabolism. Dimilin caused no significant decrease in incorporation of C’4C]lysine into the protein fraction of either sex. However, there was a significant difference in incorporation between treated and untreated weevils in the lipoprotein fraction of the male. This is something of an anomaly since we have shown earlier that the incorporation of labeled lysine appeared to precede its incorporation into protein (15), and one would expect differences occurring in one fraction to be repeated in the other. Since, however, the relative standard errors were higher in the protein fractions, they may have served to mask such a difference. ACKNOWLEDGMENTS
We wish to thank Gertrude Gibson and Betty Nail both of this laboratory for their help in oonducting the tests. REFERENCES
1. R. F. Moore, Jr. and H. M. Taft, Boll Weevils: Chemosterilization of both sexes with bu&fan plus Thompson-Hayward TH-6040, J. Econ. Entomol. 68, 96 (1975).
IN
BOLL
WEEVILS
563
2. R. Mulder and M. J. Gijswijt, The laboratory evaluation of two promising insecticides which interfere with cuticle deposition. Pestic. Sci. 4, 737 (1973). 3. L. C. Post and W. R. Vincent, A new insecticide inhibits chitin synthesis, Natllrwissenschaften 60, 431 (1973). 4. I. Ishaaye and J. E. C&da, Dietary TH-6040 alters composition and enzyme activity of housefly larvae cuticle, Pestic. Biochem. Physiol.
4, 484 (1974).
5. N. Mitlin, The physiology and toxicology of chemosterilants, in “Proceedings of the XIIth International Congress on Entomology,” London, 1964 (1965). 6. N. Mitlin and G. Wiygul, Synthesis of nucleic acid and protein in the boll weevil fed with busulfan, Bnn. Entomol. Sot. Amer. 64, 822 (1971). 7. N. Mitlin and G. Wiygul, Incorporation and metabolism of ‘%-labeled tryptophan-3 in the boll weevil Anthonomus grand& Boheman, Comp. Biochem.. Physiol. 30, 375 (1969). 8. A. C. Bartlett, Genetic markers in the boll weevil, J. Hered. 58, 159 (1967). 9. R. T. Cast, Oviposition and fecundity of boll weevils in mass-rearing cultures. J. Econ. Entomol.
59, 173 (1966).
10. W. C. Schneider, Phosphorus compounds in animal tissues. III. A comparison of methods for the estimation of nucleic acids, J. Bid. Chem. 164, 747 (1946). 11. I>. T. Mahin and R. T. Lofberg, A simplified met,hod of sample preparation for determination of tritium, carbon-14, or sulfur-35 in blood or t,issue by liquid scint,illation counting, Anal. Biochem. 16, 500 (1966). 12. G. Schmidt and S. J. Thannhauser, A method for the det,ermination of desoxyribonucleic acid, ribonucleic acid and phospho-protein in animal tissues, J. Biol. Chem. 161, 83 (1945). 13. D. B. Duncan, Multiple range and multiple F tests, Biometrics 11, 1 (1955). 14. R. W. Zumwalt, D. Roach, and C. W. Gehrke, Gas-liquid chromatography of amino acids in biological substances, J. Chromabgr. 53, 171 (1970). 1.5. N. Mitlin, G. Wiygul, and G. L. Lusk, Incorporation of lysine-6-04 int’o the protein of the adult boll weevil, Anthonomus grads, J. Insect Physiol. 14, 1277 (1968). 16. G. R. Watson and J. P. Williams, An improved method for collection and measurement of radioactivity in compounds separated by gasliquid chromatography, J. Chromatogr. 67, 221 (1972). 17, G. P. Wheeler, Some biochemical effects of dkylating agents, Fed. Proc. 26, 885 (1967).