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Laboratory studies were done to measure physiological effects of diflubenzuron when formulated in cottonseed oil and applied at very low rates to boll weevils, Anthonomus grandis grandis Boheman. Tests were designed to measure cuticle softness, mating propensity, and effect on flight. We found significant differences within treatments in all three studies as the dose increased. Cottonseed oil formulations containing 25% diflubenzuron reduced cuticle hardness by 46.2–61.0%. Cottonseed oil formulations containing 12.5–25% diflubenzuron reduced mating by 22.2–35.4% and flight by 11.6–21.2%.

Boll weevils, Anthonomus grandis grandis Boheman, dipped into solutions containing 0.25-2.0% of CIBA-GEIGY’s CGA-184699 killed >40% after 1 wk. Feeding 0.25-1.5% of this material in the adult diet for 3 d produced mortality similar to that found in tests. Female boll weevils treated with this insect growth regulator by dipping or feeding and mated with untreated males resulted in 95-99% less egg laying compared with the control. Male boll weevils administered CGA-184699 by dipping or feeding and mated with untreated females produced 58.8-73.0% fewer eggs compared with the control. Egg hatch and adult emergence of the few eggs collected was reduced to virtually zero in dipping and feeding tests.

Male boll weevils, Anthonomus grandis Boheman, sterilized with nine fractionated doses totaling 48 gray (Gy) of irradiation successfully competed with normal untreated males for virgin females. The ratios of Smale:Nmale:Nfemale tested were 1:1:1, 5:1:1, 10:1:1, 20:1:1, and 40:1:1. We determined that Smales, collectively, at a 20:1 or 40:1 ratio of Smale:Nmale were 90.8–95.1% as competitive as Nmales. A 40:1:1 ratio of Smale:Nmale:Nfemale resulted in no matings between Nmales and Nfemales alone. There were an average of 1.8 matings when both Nmale and Smales mated with the Nfemale resulting in 99.9% sterility (based on F1 adult emergence from eggs collected).

In 1992 and 1993, newly emerged adult boll weevils, Anthonomus grandis grandis Boheman, were released on diflubenzuron (DFB)-treated and untreated cotton plants grown in 20 screened field cages. Seven days later weevils were recovered, and the effects of DFB on adult survival, mating success of males, egg hatch, flight activity, and cuticle hardness were measured. DFB substantially reduced hatch, flight activity, and cuticle hardness in both years; survival apparently was reduced in 1992, but not in 1993. Mating success of treated males was unaffected in either year. The combined effect of reduced egg hatch, flight activity, cuticle hardness, and possibly survival demonstrates several ways in which DFB may reduce populations of boll weevils in the field.

Adult boll weevils, Anthonomus grandis grandis Boheman, were fed 100 ppm of diflubenzuron in their diet for 6 days, then exposed to 10 krads of acute irradiation. During that time, treated as well as control weevils were fed 2.5 or 10% sugar in the diet. After treatment, all weevils were fed cotton squares or diet plugs for 5 days. Both 2.5% sucrose-treated and control boll weevils fed cotton squares had higher locomotor activity for 3–4 of the 5 days tested. Treated and control weevils fed 10% sucrose during the treatment period followed by squares rather than diet plugs had significantly higher locomotor activity for 2 of the 5 days tested. Treated or control weevils fed 2.5% sucrose during treatment followed by squares after treatment had much higher responses than when they received diet plugs after treatment. Concentration of sugar in the diet during treatment did not affect locomotor activity for 4 of the 5 days tested where posttreatment diets were the same. Peaks of activity occurred at 2- to 4-h intervals each day.

Boll weevils, Anthonomus grandis Boheman were fed an artificial diet containing 100 ppm of diflubenzuron and either 2.5 or 10% sucrose for five days prior to an irradiation dose of 10 krads on day six. After the feeding plus irradiation treatment, the weevils were given cotton squares or artificial diet posttreatment. Treated weevils contained significantly higher levels of water than the control samples for five days posttreatment. In comparisons between the four treatments, feeding squares for the first two days posttreatment resulted in significantly higher water content in half the treatments. Similarly, when comparisons were made between the four controls it was also found that feeding squares resulted in more water in two groups tested. Sugar concentration did not influence water content in boll weevils.

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 difference in effect in lipoprotein synthesis, whereas no significant difference was demonstrated with females. Testicular growth was inhibited in some of the males. Diminishment of sexual function may therefore result in part from inhibition of biosynthesis of DNA by dimilin.

1. 1. Whole-body homogenate and hemolymph enzyme profiles of normal and γ-irradiated boll weevils were compared. 2. 2. The specific activity of homogenate lactic dehydrogenase (LDH) in treated males was significantly lower than that in normal males ( 73 ± 3 vs 104 ± 3 mU/mg protein ). 3. 3. The specific activity of homogenate acid phosphatase (AP) in treated females was significantly lower than that observed in normal females ( 89 ± 5 vs 109 ± 5 mU/mg protein ). 4. 4. Hemolymph LDH and AP showed a comparable activity pattern to homogenate LDH and AP activities. 5. 5. The homogenate LDH activity in normal males correlated well with egg production and egg hatching by females after mating with normal males. 6. 6. Low LDH activity corresponded with very low egg production and no egg hatching in irradiated males. 7. 7. LDH and AP may be monitored to assess the sterility of the boll weevil after γ-irradiation.

In the laboratory, four substituted benzamides (LY 121342 benzamide, 2-chloro- N -[[[6-[2,4-dichlorophenyl]methoxy]-3-pyridinyl]amino]carbonyl]-6-fluoro-; LY 135926 benzamide, N -[[[6-[2,4-dichlorophenylmethoxy]-3-pyridinyl]amino]carbonyl]-; triflumuron; and CGA 112913, N -[4-(3-chloro-5-trifluoromethyl-2-pyridinyl-oxy)-3,5-dichlorophenyl-aminocarbonyl]-2,6-difluoro-benzamide) were administered to adult boll weevils, Anthonomus grandis grandis Boheman, by dipping at concentrations ranging from 0.032 to 2.5% and feeding (3 d) at concentrations ranging from 0.25 to 2.0%. In dipping tests, descending order of effectiveness of these compounds in causing progeny reduction was as follows: LY 135926, triflumuron, LY 121342, and CGA 112913. In feeding tests, descending order of effectiveness was as follows: CGA 112913, LY 121342, triflumuron, and LY 135926. Mortality was higher in dip and feeding tests with LY 121342 and LY 135926. Although all treatments reduced flight compared with the control, boll weevils that were fed these chemicals (except LY 121342) usually flew more than those that were dipped. A few days after treatment, flight of treated and controls was reduced compared with earlier flights.

Male Anthonomus grandis Boheman fed 0.08% busulfan + 0.4% hempa for 6 days were as competitive as untreated males about ½ the time (occurripg randomly), and ½ the time the treated males were ¼–⅓% as competitive as untreated males. After an average of 7 matings, the treated males had depleted their supply of sperm. Determinations of sterility based on testes examination were in full agreement 91% of the time with determinations based on egg hatch and adult emergence.

Boll weevils, Anthonomus grandis grandis Boheman, 9 days after egg implantation in the larval diet were transported from the Boll Weevil Research Laboratory, Mississippi State, MS, to the Comparative Animal Research Laboratory, Oak Ridge, TN, and irradiated with 6.9 krad (test 1) or 7.2 krad (test 2) of /sup 60/Co gamma rays delivered in 25 equal doses over 100 h. In test 1, from 600 individual pairs of T (treated) males x N (normal) females, only 114 eggs hatched from a sample of 950 eggs, and 47 adults emerged from a sample of 1042 eggs. Also, from 600 pairs of T females x N males, 6 eggs hatched of a sample of 6 eggs and 12 adults emerged from a sample of 20 eggs. In test 2, from 700 individual pairs of T males x N females, 54 eggs hatched from a sample of 1510, and 10 adults emerged from a sample of 1703 eggs. Also, in T females x N males matings, 1 egg hatched of a sample of 3, and no adults emerged from a sample of 4. Transportation and handling in the 2nd test reduced adult emergence an avg of 49%. Thus the 2 replicates in testmore » 2 resulted in 3.4 x 10/sup 5/ and 4.3 x 10/sup 5/ irradiated weevils emerging/day for 7 days. Bacterial contamination of weevils was low.« less

Previously, boll weevils, Anthonomus grandis Boheman, were dipped in chemosterilant solutions using 100% methanol, ethanol, or acetone. It was suspected that pure solvents may often have adverse effects. Boll weevils at 5 or 6 days old, dipped in 75 to 100% methanol, ethanol, or acetone, had significantly reduced flight activity. Boll weevils dipped in 25% suspensions of methanol, ethanol, or acetone containing 0.025% diflubenzuron and then irradiated had acceptable flight activity, mortality, and sperm transfer, but the females were not adequately sterilized. Male weevils dipped in 50% methanol or ethanol suspensions containing 0.05% diflubenzuron + 10 krads of irradiation had normal flight for 4 days, were adequately sterilized and transferred sperm to more females 7 days after treatment than males dipped in acetone.

Adult Anthonomus grandis grandis Boheman were sterilized when fed an adult diet containing 100 ppm of diflubenzuron for 5 days followed by 10 krads of acute irradiation on day 6. The diet also contained either 10% sucrose, 10% maltose, or 2.5% sucrose diet (wt/wt) as a standard comparison. Mortality of treated (T) males and females fed 10% sucrose was significantly lower than T weevils fed the standard 2.5% sucrose diet for the first 4 days after treatment. Treated males fed 10% maltose also had significantly less mortality for 4 days compared with males fed the standard sucrose diet. For the first 4 days after treatment, sperm transfer of T males fed the 10% maltose diet was significantly higher than those fed the 2.5% sucrose diet. Sperm transfer of T males fed 10% sucrose was significantly higher for 2 days after treatment compared with T males fed the 2.5% sucrose diet. Treated males fed 10% maltose flew significantly more at day 1 after treatment than T males fed the other two sugar diets. Increased sugar in the diet had no overall effect in increasing male attractiveness to virgin females.

Fractionated doses of 6,250–8,000 rads of gamma irradiation when administered to pupae of the boll weevil, Anthonomus grandis Boh., sexually sterilized both sexes. Mortality of males thus treated with 6,250 and 8,000 rads via fractionation was 14% and 27% respectively, by 5 days posttreatment compared with 46% mortality when an equivalent acute dose was administered to newly emerged adults. Pheromone production of males irradiated at 6,250 rads was one-third that of the control for the first 4 days, but equal that of the control during 5–11 days posttreatment. This procedure lends itself to the large-scale sterilization of weevils needed in an eradication program. This technique is applicable to other insects that are highly susceptible to acute doses. ZUSAMMENFASSUNG STERILISATION VON BAUMWOLLKAPSELKAFERPUPPEN MIT FRAKTIONIERTEN DOSEN EINER GAMMABESTRAHLUNG Durch fraktionierte Dosen von insgesamt 6250 und 8000 rads einer Gammabestrahlung (25 bzw. 32 Teilbehandlungen zu 250 rads mit 4 h-Intervallen) wurden Puppen des Baumwollkapselkafers, Anthonomus grandis Boheman, in beiden Geschlechtern sexuell sterilisiert. Die Sterblichkeit, 5 d nach der Behandlung, betrug bei den so behandelten Mannchen bei 6250 rads 14%, bei 8000 rads 27% im Vergleich zu 46%, wenn eine aquivalente akute Dosis bei frisch geschlupften Kafern angewendet wurde. Die Pheromonproduktion der Mannchen in den ersten 4 d erreichte bei 6250 rads-Bestrahlung 1/3 derjenigen der Kontrolltiere; 5–11 d nach der Behandlung war kein Unterschied. Dieses Verfahren eignet sich fur die Massensterilisation von Kapselkafern, wie sie bei einem Ausrottungsprogramm notig ist. Diese Technik ist anwendbar auf andere Insekten, die gegenuber akuten Dosen hochempfindlich sind.

When 6–7-days old mass-reared ebony boll weevils, Anthonomus grandis Boheman, were given 25 doses of γ-irradiation totaling 6625 rads, mortality was 14%–15% less 1 week later than when young weevils (1–2 day-old) were similarly treated. However, giving older weevils an acute dose of 6625 rads did not reduce mortality. Seven-day-old weevils receiving the acute treatment mated 10% more than weevils that were 3 days old at the time of treatment. Seven-day-old male weevils exposed to the fractionated treatment transferred sperm to 12% more females than 7-day-old males exposed to the acute treatment. ZUSAMMENFASSUNG FRAKTIONIERTE UND EINMALIGE BESTRAHLUNG: DIE FOLGEN DER ALTERUNG ADULTER ANTHONOMUS GRANDIS VOR DER BEHANDLUNG Adulte Anthonomus grandis Boheman aus einer Massenzucht wurden einer fraktionierten Behandlung mit 25 Portionen γ-Strahlung von total 6625 rad unterworfen. Wenn gealterte Kafer von 6–7 Tagen bestrahlt wurden, war ihre Mortalitat 1 Woche spater 14%–15% geringer als bei Kafern, die mit 1–2 Tagen behandelt worden waren. Wenn die Bestrahlung in einer einmaligen Dosis von 6625 rad erfolgte, war das Alter der Tiere ohne Bedeutung. Kafer, die mit 7 Tagen eine fraktionierte oder eine einmalige Behandlung erhalten hatten, kopulierten nur 9% und 10% sooft wie Kafer, welche dieselbe Behandlung mit 3 Tagen erhalten hatten. Kafer, die mit 7 Tagen fraktioniert behandelt worden waren, ubermittelten Spermien an 12% mehr Weibchen als Kafer, die mit 7 Tagen einmalig bestrahlt worden waren.

Irradiation (10 krads in a nitrogen atmosphere) plus dipping in 0.1% diflubenzuron in acetone of 6-day-old Anthonomus grandis grandis Boheman was selected over fumigation plus dipping of adults and fractionated irradiation of pupae as the method of choice for sterilization for production of quality weevils. The results for this method were: sterility 98.5% mortality 34.5% at 7 days posttreatment; mating with sperm transfer, 78.5% and 59.6%, of normal at 3 and 7 days posttreatment, respectively; and pheromone production of 0.4 µg/adult/day. Fumigation plus pentluron produced comparable sterile males, but irradiation was more convenient and safer.

Boll weevils, Anthonomus grandis grandis Boheman, were sterilized by dipping in an aqueous suspension containing 0.4% (AI) diflubenzuron and antibiotics (0.03% kanamycin, 0.015% penicillin, and 0.015% chloramphenicol) for 10 min followed by 10 krad of irradiation. Overall, mortality was ≤16.6% after 5 d; by 7 d, male mortality was ≤59.5%. Treated males were 77–79% as attractive as untreated males to untreated females. Flight activity of treated weevils was 45–56% of that observed in the control. In competitive mating studies, numbers of females mated by untreated males were significantly greater than treated males at a ratio of 1:1. When treated males outnumbered untreated males by more than 5:1, the number of matings to untreated females was significantly higher. A 20:1 ratio of treated males to untreated males was required to significantly reduce F1 adult emergence. Sterility, mortality, and vigor were increased with this new method compared with the previous method that involved feeding diflubenzuron (100 ppm) for 5 d followed by 10 krad of irradiation.

The standard method of sterilizing Anthonomus grandis Boheman (6-day feeding of 0.1% busulfan, 1-4-butanediol dimethanesulfonate) adequately sterilizes males but does not induce a high degree of sterility in females, and mortality is less than 40% after 2 weeks. Feeding higher concentrations of busulfan alone or lower concentrations of busulfan in combinations with methotrexate, hempa, or apholate for 6 days or less, or feeding 0.1-0.2% busulfan followed by low doses of gamma radiation induces virtually complete sterility in both sexes but causes fairly high mortality. A 4-day feeding of 0.2% busulfan + 0.25% methotrexate resulted in complete sterility in females and almost complete sterility in males, and mortality was less than 40% by 3 weeks after treatment.

A device, consisting of two 5-cm cube boxes with screen tops and cone-shaped screens opening into the inside, was used to measure the attractiveness of the male Anthonomus grandis Boheman contained inside the 5-cm cube boxes in a screen-covered bottle cap above the screen cone opening. The small boxes were placed inside an enclosed cube boxes in a screen-covered bottle cap above the screen cone opening. The small boxes were placed inside an enclosed plastic box (26 by 35 by 8 cm) provided with a top to contain the loose females. Tests were run for 24 h. Sterile males used in some of the tests had been fed for 5 days on an artificial diet containing diflubenzuron at 100 ppm, followed by a 10-krad acute dose in a 137Cs irradiator. When sterile males were tested against normal males for female responsiveness, the normal males were 1.6 times more attractive for the first 5 days after treatment.

When pupae of Anthonomus grandis grandis Boheman were fumigated for 5 h in a circulating, saturated atmosphere of bisazir, 99% of the males were sterilized, and egg laying by treated females outcrossed with normal weevils was reduced by 90.6%. Sterile males transfered 11.8% more sperm than control males the 1st week post treatment but 42.6% less the 2nd week.

Male boll weevils, Anthonomus grandis Boheman, reared in the laboratory and sterilized with busulfan (1,4-butanediol dimethan-sulfonate) alone were more attractive to ♀ weevils than those treated with combinations of busulfan and hempa. In general, sterilization reduced the attractiveness of laboratory-reared males by ca. 50%. Evidence is presented that shows a great range of variability in the attractiveness of males of 1 type. These sterilized ♂ boll weevils are as attractive to ♀ weevils as overwintered field males.

Overwintered Anthonomus grandis Boheman can be sterilized and egg laying can be greatly reduced in the laboratory by feeding them a diet containing busulfan (1,4-butanediol dimethanesulfonate). A feeding table designed to attract weevils as they emerged from hibernation quarters attracted a small percentage of the total overwintered weevils that were in the field on July 1.

Cotton squares containing an egg deposited by Anthonomus grandis Boheman were significantly denser than cotton squares that did not contain an egg. This increased density may cause mortality of the egg stage.

After virgin matings with sterile males (previously mated 0 to 3 times) the responsiveness of females of Anthonomus grandis Boheman to grandlure was twice that of females mated with normal males. The higher responsiveness of females mated with sterile males indicate 0 to 66 percent fewer sperm were transferred to the spermathecae and these females continued seeking other mates. After 3 matings, sperm transferred by /sup 60/Co irradiated males was reduced by 67 percent or more.

Laboratory reared adults (25 newly emerged, mixed sexes) were immersed in acetone solutions of the candidate chemicals for 5 sec, air dried, and held at 27°C and 60% RH, fresh field squares (cotton buds) were then provided daily (1 square/5 weevils) for feeding and oviposition. Also, the candidate chemicals were placed in the adult diet (0.5%), and newly emerged adults were allowed to feed for 3 days. Eggs from both treatments were collected; one-half the eggs were held for hatch, and one-half the eggs were placed on larval diets for determination of adult emergence. High adult mortality was obtained with chemicals JURD 2693 and JURD 2581 when incorporated within the diet. Chemical JURD 2335 in the diet also gave a reduced adult emergence. None of the compounds were active when tested by dipping in acetone solution.

Adult boll weevils (reared in the laboratory) were tested by dipping (4% or less in acetone solution) in the candidate chemical for 5 sec, air dried, and held at 30°C and 40 to 50% RH. Ten diet pellets were provided fresh daily for food and ovipositing. The candidate chemicals were also placed in the adult diet (0.5 or 0.25%) and fed to the newly eclosed weevils for 3 days. Eggs collected at 7 and 14 days posttreatment were divided and half held (2 weeks) for adult PI emergence. Three hundred and fourteen candidate chemicals were tested by feeding of which 13 were further tested by dipping.