J. Med.
4 September 1979
Entomol. Vol. 15, nos. 5-6: 506-509 ©
1978 by
the Bishop Museum
LABORATORY AND FIELD EVALUATION OF A GROWTH-REGULATING COMPOUND (TH-6040) AGAINST CULEX PIPIENS FATIGANS (DIPTERA: CULICIDAE)! By V. P. Sharma2, C. P. Batra3 and G. D. Brooks4
dysis and this results in death (Van Daalen et al. 1972, Wellinga et aI. 1973, Post & Vincent 1973). The data presented by the manufacturer show that the compound must be ingested continuously through at least 1 instar before mortality occurs and can be assessed (Philips-Dupher 1972).
This paper reports the results of a series of experiments carried out to investigate the biological effects of TH-6040 on immatures and adults of Culex pipiens fatigans Weid. The possibility that IGR compounds may induce sterility in surviving adults was also investigated. As part of this assessment, the sterilizing potential of the chemical and its effect on female fecundity were evaluated, as well as the effects on mating ability of the males. As this chemical is a known growth inhibitor, studies also were carried out on the sensitivity of immature stages to chemical treatment, with followup studies on morphological abnormalities in the imago after treatment. To determine application dosages to achieve control in the field, TH-6040 was sprayed in drains serving as breeding sites for Cx. p. fatigans.
Recent research has led to the discovery of a new chain of chemical compounds which act as insect growth regulators (IGR), including insect juvenile hormones and their mimics. A number of such growth regulators have now become available for experimentation. Jakob & Schoof (1971, 1972),Jakob (1972, 1973), Schaefer & Wilder (1972), Steelman & Schilling (1972) and Mulla et al. (1974) have evaluated the efficiency of a number of these materials against mosquitoes. Miura & Takahashi (1973, 1974) also evaluated the effect of certain of these insect growth regulators on nontarget aquat. . 1C orgaOisms. MATERIALS AND METHODS The growth-regulating compound5 TH-6040 All laboratory experiments were carried out un[=OMS-1804 or Dimilin, i.e., 1-(4-chlorophenyl)der controlled conditions of temperature (29° ± 3-(2,6-difluorobenzoyl)-ureaJ does not interfere with the hormone regulation of the molting pro- 2°C) and humidity (80% ± 10% RH), using Cx. p. cess. The mode of action is interference in cuticle fatigans (Delhi strain) as the test insect. Test insects formation, which induces difficulties in molting. for laboratory studies were reared by the method As a result, immature stages cannot withstand the described by Singh et al. (1975). As TH-6040 was made available as a 25% wettable powder, all diincreased turgor and/or muscle traction during eclutions for this study were made in water. To establish LC95 values for this compound, late 3rd- or early 4th-instar larvae were exposed for 24 'WHO/ICMR Research Unit on Genetic Control of Mosqui- h in TH-6040 concentrations in distilled water toes, New Delhi, India. without food, following the standard World 2Malaria Research Centre, 22 Sham Nath Marg, Delhi· Health Organization insecticide susceptibility 110054, India. 3Vector Control Research Cemre, Kosapalayam, Pondichermethod (1970), except for (I) mortality counts, ry-6050 II, India. which were made at emergence for determination 4Vector Biology and Control Division, Bureau of Tropical of LC50 and LC95 values, and (2) treated larvae subDiseases, Atlanta, Georgia 30333, USA. 5TH-6040 has an oral LD50 of 10,000 mg/kg weight of rats sequently washed and reared in the conventional (manufacturer's data). manner in the presence of food. In this procedure,
Downloaded from http://jme.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on October 31, 2015
Abstract: A growth-regulating compound (TH-6040) was evaluated for the comrol of Culex pipiens fatigans. Applied at the LC., dosage (0.001 ppm), the compound did not induce sterility in males or females or affect the mating ability of treated males. The compound decreased the life span of both sexes surviving after treatment; this effect was less pronounced when sexes were caged together. Third-instar larvae were more sensitive than 4th-instar larvae, and pupae showed no adverse effects in response to treatment. However, TH-6040 induced many morphological abnormalities which were visible in emerged adults. Eighty to 100% comrol of ex. p.fatigans breeding in polluted drains was achieved by application to larvae in target dosages of 0.5 ppm and 1.0 ppm. The residual effect of this larvicide in field applications was approximately 4 days.
1979
Sharma
et al.: Growth-regulating
Development
of
morPhological
abnormalities.
against
Culex p. fatigans
507
TABLE 1. Percentage sterility in d and '( Cx. p.fatigans treated as larvae in 0.00 I ppm (a.i.) TH-6040. STERILITY TEST
I
2 3 4
(%)
CROSSES
Normal NOI'mal Treated Treated
0 0 d 0
x x x x
Normal Treated Normal Treated
'( '( '( '(
1.04
1.06 1.02 1.01
cage was recorded daily and the results tabulated on the basis of cumulative percentage mortality at 5-day intervals. Fecundity of treated females. Egg rafts from the survival studies were collected for 4 successive gonotrophic cycles and the number of blood-fed females in each cycle was recorded. These data were used to calculate the percentage oviposition. Sensitivity of immature stages. To test the sensitivity of 3rd- and 4th-instar larvae and pupae to TH6040, the respective stages (100 each, replicated 5 times) were held in a 0.00 I ppm insecticide concentration. The larvae were removed after pupation and held in water for eclosion, whereas pupae were held in the insecticide solution until eclosion. A daily record of mortality was maintained to calculate the survival of the immatures.
Observations were made at 24-h intervals during larval exposure through emergence of adults. All morphological abnormalities in immatures and adults were recorded. Sterility in adults. Adults were mass-mated in Determination of insecticide aPPlication dosages for laboratory cages using 100 males and 100 females control of breeding in drains. Larvicidal treatment (replicated 5 times). After cohabiting for 5 days, using TH-6040 25% wettable powder was applied the females were offered a blood meal. Ten egg in polluted effluent drains. A compression sprayer rafts from each cage were randomly selected and was used to spray selected drains at target dosages examined under the microscope to calculate the of 0.1,0.25,0.5 and 1.0 ppm active ingredient. All tests were carried out in heavily polluted drains, percentage sterility. which were unlikely to be used by animals as a Mating ability of males. Males surviving treatment were caged with virgin females (1 male:6 fe- source of drinking water. Drains with water movemales) for a period of 5 days. Each experiment was ment were avoided. The density of immatures was replicated 10 times. Females were checked for in- estimated on the basis of the dipper sampling method using 3 standard dips per 10 linear meters semination by examination of the spermathecae of drain. The average per dip of all stages of larvae under the microscope. Longevity of treated adults. For studies on the and pupae was recorded. Observations on density longevity of emerged adults, 100 treated or un- were made prior to spraying and daily thereafter treated males or females were held separately in until a new brood of 2nd- and 3rd-instar larvae laboratory cages. Each test was replicated 4 times. reappeared in the drain. In another test series, 4 types of crosses were made, as follows: (1) normal males X normal feRESULTS males; (2) normal males x treated females; (3) LC50 and LC95 values for immatures of Cx. p. fatitreated males x normal females and (4) treated gans. Late 3rd- or early 4th-instar larvae were exmales x treated females. Each test was replicated 5 times. Adults were offered 1% glucose on cotton posed to TH-6040 concentrations, and results are pads and females were offered a blood meal on based on 3 sets of 5 replicates. The LCso for 24-h 2-day-old chicks for 4 successive gonotrophic cycles. exposure (without food) is 0.003 ppm (a.i.) and for The number of mosquitoes found dead in each continuous exposure (with food) is 0.0005 ppm
Downloaded from http://jme.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on October 31, 2015
live pupae were transferred daily to untreated water in plastic cups for further observations, i.e., normal emergence, presence of morphological abnormalities or death. Partially emerged adults or those found completely emerged but unable to leave the water surface were recorded separately and scored as dead. In a 2nd procedure, larvae were reared under conditions which provided a continuous food supply and continuous exposure to the chemical. In each test, 25 fourth-instar larvae were held in 250 ml of test suspension in a 500-ml beaker. Each test was replicated 10 times with concurrent controls. Test suspensions were prepared in distilled water to give a dosage in parts per million of the active ingredient. Mortality of the larvae and pupae was recorded daily. To study the possible effects on treated insects, 4th-instar larvae were treated continuously until pupation (with food) in 0.00 I ppm of active ingredient (a.i.) TH-6040 solution. Ensuing adults were held in 30 X 30 X 30 cm laboratory cages and used in the following studies.
compound
J.
508
Med. Entomol.
TABLE2. Cumulative % adult mortality of ex. p.fatigans treated as larvae in 0.001 ppm (a.i.) TH-6040.
TABLE4. Survival of 3rd- and 4th-instal' larvae and pupae of Cx. p.fatigans exposed to 0.001 ppm (a.i.) TH-6040.
AVERAGE% MORTALITY FOR 5-DAYPERIODSAT INDICATED INTERVAL AFTEREMERGENCE
o and MOSQUITO TEST CROSSES
o Normal
o and
4 September 1979
Entomol. Vol. 15, nos. 5-6: 506-509 ©
1978 by
the Bishop Museum
LABORATORY AND FIELD EVALUATION OF A GROWTH-REGULATING COMPOUND (TH-6040) AGAINST CULEX PIPIENS FATIGANS (DIPTERA: CULICIDAE)! By V. P. Sharma2, C. P. Batra3 and G. D. Brooks4
dysis and this results in death (Van Daalen et al. 1972, Wellinga et aI. 1973, Post & Vincent 1973). The data presented by the manufacturer show that the compound must be ingested continuously through at least 1 instar before mortality occurs and can be assessed (Philips-Dupher 1972).
This paper reports the results of a series of experiments carried out to investigate the biological effects of TH-6040 on immatures and adults of Culex pipiens fatigans Weid. The possibility that IGR compounds may induce sterility in surviving adults was also investigated. As part of this assessment, the sterilizing potential of the chemical and its effect on female fecundity were evaluated, as well as the effects on mating ability of the males. As this chemical is a known growth inhibitor, studies also were carried out on the sensitivity of immature stages to chemical treatment, with followup studies on morphological abnormalities in the imago after treatment. To determine application dosages to achieve control in the field, TH-6040 was sprayed in drains serving as breeding sites for Cx. p. fatigans.
Recent research has led to the discovery of a new chain of chemical compounds which act as insect growth regulators (IGR), including insect juvenile hormones and their mimics. A number of such growth regulators have now become available for experimentation. Jakob & Schoof (1971, 1972),Jakob (1972, 1973), Schaefer & Wilder (1972), Steelman & Schilling (1972) and Mulla et al. (1974) have evaluated the efficiency of a number of these materials against mosquitoes. Miura & Takahashi (1973, 1974) also evaluated the effect of certain of these insect growth regulators on nontarget aquat. . 1C orgaOisms. MATERIALS AND METHODS The growth-regulating compound5 TH-6040 All laboratory experiments were carried out un[=OMS-1804 or Dimilin, i.e., 1-(4-chlorophenyl)der controlled conditions of temperature (29° ± 3-(2,6-difluorobenzoyl)-ureaJ does not interfere with the hormone regulation of the molting pro- 2°C) and humidity (80% ± 10% RH), using Cx. p. cess. The mode of action is interference in cuticle fatigans (Delhi strain) as the test insect. Test insects formation, which induces difficulties in molting. for laboratory studies were reared by the method As a result, immature stages cannot withstand the described by Singh et al. (1975). As TH-6040 was made available as a 25% wettable powder, all diincreased turgor and/or muscle traction during eclutions for this study were made in water. To establish LC95 values for this compound, late 3rd- or early 4th-instar larvae were exposed for 24 'WHO/ICMR Research Unit on Genetic Control of Mosqui- h in TH-6040 concentrations in distilled water toes, New Delhi, India. without food, following the standard World 2Malaria Research Centre, 22 Sham Nath Marg, Delhi· Health Organization insecticide susceptibility 110054, India. 3Vector Control Research Cemre, Kosapalayam, Pondichermethod (1970), except for (I) mortality counts, ry-6050 II, India. which were made at emergence for determination 4Vector Biology and Control Division, Bureau of Tropical of LC50 and LC95 values, and (2) treated larvae subDiseases, Atlanta, Georgia 30333, USA. 5TH-6040 has an oral LD50 of 10,000 mg/kg weight of rats sequently washed and reared in the conventional (manufacturer's data). manner in the presence of food. In this procedure,
Downloaded from http://jme.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on October 31, 2015
Abstract: A growth-regulating compound (TH-6040) was evaluated for the comrol of Culex pipiens fatigans. Applied at the LC., dosage (0.001 ppm), the compound did not induce sterility in males or females or affect the mating ability of treated males. The compound decreased the life span of both sexes surviving after treatment; this effect was less pronounced when sexes were caged together. Third-instar larvae were more sensitive than 4th-instar larvae, and pupae showed no adverse effects in response to treatment. However, TH-6040 induced many morphological abnormalities which were visible in emerged adults. Eighty to 100% comrol of ex. p.fatigans breeding in polluted drains was achieved by application to larvae in target dosages of 0.5 ppm and 1.0 ppm. The residual effect of this larvicide in field applications was approximately 4 days.
1979
Sharma
et al.: Growth-regulating
Development
of
morPhological
abnormalities.
against
Culex p. fatigans
507
TABLE 1. Percentage sterility in d and '( Cx. p.fatigans treated as larvae in 0.00 I ppm (a.i.) TH-6040. STERILITY TEST
I
2 3 4
(%)
CROSSES
Normal NOI'mal Treated Treated
0 0 d 0
x x x x
Normal Treated Normal Treated
'( '( '( '(
1.04
1.06 1.02 1.01
cage was recorded daily and the results tabulated on the basis of cumulative percentage mortality at 5-day intervals. Fecundity of treated females. Egg rafts from the survival studies were collected for 4 successive gonotrophic cycles and the number of blood-fed females in each cycle was recorded. These data were used to calculate the percentage oviposition. Sensitivity of immature stages. To test the sensitivity of 3rd- and 4th-instar larvae and pupae to TH6040, the respective stages (100 each, replicated 5 times) were held in a 0.00 I ppm insecticide concentration. The larvae were removed after pupation and held in water for eclosion, whereas pupae were held in the insecticide solution until eclosion. A daily record of mortality was maintained to calculate the survival of the immatures.
Observations were made at 24-h intervals during larval exposure through emergence of adults. All morphological abnormalities in immatures and adults were recorded. Sterility in adults. Adults were mass-mated in Determination of insecticide aPPlication dosages for laboratory cages using 100 males and 100 females control of breeding in drains. Larvicidal treatment (replicated 5 times). After cohabiting for 5 days, using TH-6040 25% wettable powder was applied the females were offered a blood meal. Ten egg in polluted effluent drains. A compression sprayer rafts from each cage were randomly selected and was used to spray selected drains at target dosages examined under the microscope to calculate the of 0.1,0.25,0.5 and 1.0 ppm active ingredient. All tests were carried out in heavily polluted drains, percentage sterility. which were unlikely to be used by animals as a Mating ability of males. Males surviving treatment were caged with virgin females (1 male:6 fe- source of drinking water. Drains with water movemales) for a period of 5 days. Each experiment was ment were avoided. The density of immatures was replicated 10 times. Females were checked for in- estimated on the basis of the dipper sampling method using 3 standard dips per 10 linear meters semination by examination of the spermathecae of drain. The average per dip of all stages of larvae under the microscope. Longevity of treated adults. For studies on the and pupae was recorded. Observations on density longevity of emerged adults, 100 treated or un- were made prior to spraying and daily thereafter treated males or females were held separately in until a new brood of 2nd- and 3rd-instar larvae laboratory cages. Each test was replicated 4 times. reappeared in the drain. In another test series, 4 types of crosses were made, as follows: (1) normal males X normal feRESULTS males; (2) normal males x treated females; (3) LC50 and LC95 values for immatures of Cx. p. fatitreated males x normal females and (4) treated gans. Late 3rd- or early 4th-instar larvae were exmales x treated females. Each test was replicated 5 times. Adults were offered 1% glucose on cotton posed to TH-6040 concentrations, and results are pads and females were offered a blood meal on based on 3 sets of 5 replicates. The LCso for 24-h 2-day-old chicks for 4 successive gonotrophic cycles. exposure (without food) is 0.003 ppm (a.i.) and for The number of mosquitoes found dead in each continuous exposure (with food) is 0.0005 ppm
Downloaded from http://jme.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on October 31, 2015
live pupae were transferred daily to untreated water in plastic cups for further observations, i.e., normal emergence, presence of morphological abnormalities or death. Partially emerged adults or those found completely emerged but unable to leave the water surface were recorded separately and scored as dead. In a 2nd procedure, larvae were reared under conditions which provided a continuous food supply and continuous exposure to the chemical. In each test, 25 fourth-instar larvae were held in 250 ml of test suspension in a 500-ml beaker. Each test was replicated 10 times with concurrent controls. Test suspensions were prepared in distilled water to give a dosage in parts per million of the active ingredient. Mortality of the larvae and pupae was recorded daily. To study the possible effects on treated insects, 4th-instar larvae were treated continuously until pupation (with food) in 0.00 I ppm of active ingredient (a.i.) TH-6040 solution. Ensuing adults were held in 30 X 30 X 30 cm laboratory cages and used in the following studies.
compound
J.
508
Med. Entomol.
TABLE2. Cumulative % adult mortality of ex. p.fatigans treated as larvae in 0.001 ppm (a.i.) TH-6040.
TABLE4. Survival of 3rd- and 4th-instal' larvae and pupae of Cx. p.fatigans exposed to 0.001 ppm (a.i.) TH-6040.
AVERAGE% MORTALITY FOR 5-DAYPERIODSAT INDICATED INTERVAL AFTEREMERGENCE
o and MOSQUITO TEST CROSSES
o Normal
o and
