Effects of Pyriproxyfen on Engorged Females and Newly Oviposited ...

Effects of Pyriproxyfen on Engorged Females and Newly Oviposited Eggs of the Lone Star Tick (Acari: Ixodidae) P. D. TEEL, W. A. DONAHUE, O. F. STREY, AND R. W. MEOLA Department of Entomology, Texas A&M University, College Station, TX 77843-2475

J. Med. Entomol. 33(5): 721-725 (1996) ABSTRACT Engorged females and 1- to 3-d-old eggs of the lone star tick, Amblyomma americanum (L.), were exposed to 9 treatments of pyriproxyfen (4, 8, and 16 /Ag/cm2) in glass vials at exposure periods of 7 d, 14 d, and continuous at each dosage level. Treatment of newly engorged females did not affect the number of females ovipositing, but the number of eggs oviposited decreased as dosage and exposure time increased. Complete inhibition of egg hatch occurred at all treatment levels except that of the lowest dosage and exposure time where 99.9% inhibition was observed. Egg masses from treated females contained eggs that turned a dark amber color and imploded, retained normal shape and color without visible evidence of embryogenesis, or had developed embryos that were unable to emerge. Treatments of 1to 3-d-old eggs were effective in reducing hatch and larval survivorship to 25 d after emergence only at the continuous exposures of dosages of 4, 8, and 16 /ig/cm2. Larvae emerging from eggs treated at the lower dosage rates of 0.2 and 0.02 ^tg/cm2 were as successful in feeding on chickens and subsequently molting as acetone treated control ticks. KEY WORDS juvenile hormone analog, ovicide, fecundity, ixodidae, pyriproxyfen

have successfully been used as ovicidal agents for cat flea control (Meola et al. 1993) on both cats and dogs. Pyriproxyfen, 2-[l-methyl-2-(4-phenoxyphenoxy) ethoxy] pyridine, is one of the latest juvenile hormone analogs being developed as an ectoparasiticide for on-animal application because of its high degree of insecticidal activity, environmental stability, and safety to mammals. One of the deficiencies of juvenile hormone analogs is their limited activity on noninsect ectoparasites such as ticks and mites. Past investigations have demonstrated some activity of the juvenile hormone analogs to various developmental stages of hard ticks, but no single compound has provided the degree of activity necessary for commercial development. Treatment of newly engorged Boophilus decoloratus (Koch); southern cattle tick, B. microplus (Canestrini); and Amblyomma hebraeum Koch with juvenile hormone analogs caused desiccation of eggs, and the number of affected eggs was dose dependent (Solomon and Evans 1977). Hatch of eggs from Hyalomma dromedarii Koch and American dog tick, Dennacentor variabilis (Say), was reduced after topical treatment on the 1st d of oviposition with 3 juvenile hormone analogs (Bassel 1974, McDaniel and Oliver 1978). Application technique of the various compounds may account for some of the variation noted in the literature. In general, the juvenile hormone analogs were more active when injected than with topical application (Mansingh and Rawlins 1977). This report JUVENILE HORMONE ANALOGS

demonstrates ovicidal activity in the lone star tick, Amblyomma americanum (L.), when exposed to surface residues of pyriproxyfen. Materials and Methods Technical pyriproxyfen 97.0% (AI) was provided by McLaughlin Gormley King (Minneapolis, MN). Acetone used for dilutions was dried over anhydrous sodium sulfate to remove any water from the acetone. The following stock solutions were prepared in volumetric flasks: (A) 93.28 mg (AI)/250 ml acetone, (B) 186.56 mg (AI)/250 ml acetone, (C) 373.12 mg (AI)/250 ml acetone, and (D) acetone only. An aliquot of stock solution was pipetted into a glass vial laid on its side. The vial was then rolled to evenly coat the inner surface. Vials were rolled several times until the acetone was evaporated and allowed to remain in a chemical hood for 6-8 h to thoroughly dry. Acetone controls were also treated in the same manner. All vials were prepared on the same day for all treatments and the treated and control vials were placed back in the original boxes and stored at — 15°C until needed. These procedures were not expected to alter the resulting concentration of pyriproxyfen on the glass surface because of the high stability of the material over the range of temperature exposures ( — 1530°C), stability in organic solvents including acetone, and low volatility (MGK Technical Bulletin 1991).

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Engorged Female Bioassay. The bioassay was conducted using 3.0-dram vials that had been washed in hot soapy water, rinsed in distilled water, and placed into a drying oven at >205°C overnight. The vials were subsequently treated with 0.30 ml of stock solutions A, B, and C to provide 4.0, 8.0, and 16.0 /Ag/cm2 of pyriproxyfen, respectively. Ticks used in these evaluations were from a colony of A. americantim originating from Brazos County, Texas, and maintained at the Tick Research Laboratory, Department of Entomology, Texas A&M University, College Station. Adult A. americantim were fed at a sex ratio of 1:1 on stanchioned bovines in stockinette cells. Engorged female ticks were placed randomly in treatments within 24—72 h after detachment. Vials containing ticks were capped and covered with fine mesh cloth and held in an environmental chamber at ~30°C, 70-80% RH, and a photoperiod of 14:10 (L:D) h. To establish 7- and 14-d exposures at each dosage level, engorged ticks and any eggs were transferred to untreated vials based on the date they were placed initially into the treated vials. Ticks exposed continuously were left in their original vials for the duration of the study. The engorged females were scored for oviposition. Eggs were scored for condition of unhatched eggs and larval hatch. Egg Bioassay. This bioassay was conducted with 2.0-dram vials prepared in the same manner as described previously except that each vial was treated with 0.25 ml of stock solution calculated for the smaller internal surface area. In addition to the standard treatment concentrations of 4.0, 8.0, and 16.0 /Ltg/cm2 of pyriproxyfen, 3 lower concentrations were also evaluated. The lower concentrations of 2.0, 0.2, and 0.02 /ng/cm2 pyriproxyfen were prepared as described previously. The lower concentrations were evaluated because results from the engorged female bioassay indicated a high degree of activity at all 3 of the concentrations tested. Mated engorged female ticks were obtained by feeding adults at a sex ratio of 1:1 on cattle in orthopedic stockinette cells. Engorged ticks were each placed in untreated glass vials, held at 30°C, 85% RH, a photoperiod of 14:10 (L:D) h, and monitored for oviposition. Once oviposition began, eggs were harvested at 3-d intervals over no more than a 15-d oviposition period. Same-aged eggs were pooled into a single petri dish and gently mixed to homogenize each collection of eggs. Eggs were handled with waxed paper scoopulas to minimize physical damage. The treated vials were warmed to room temperature before eggs were placed into them. Twenty (20) replicates of each dosage-exposure time were prepared except for the acetone control for which only the continuous exposure time was evaluated. A small aliquot of —250-300 eggs was placed into each vial to give approximately equal numbers of eggs to each replicate. The vials were covered with a double piece of fine mesh cloth, secured with a screw cap with

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a hole in it, and placed in the environmental chamber 30°C, 70-80% RH, and a photoperiod of 14: 10 (L:D) h. The egg assay was scored for percentage of hatch, percentage of larvae alive, and the number of white fecal spots. The percentage hatch and percentage of live larvae were estimated to within 5% increments, and the number of fecal spots were estimated to the nearest 25 spots. Estimates of fecal spots were based on counting 1/5 of the circumference from vial top to bottom as a density gradient of spots existed along the vial length (vials were maintained upright during the trial). Total spots were estimated by multiplying by 5. Each vial was counted individually using this procedure. A dissecting microscope objective was set to 1.5X, and the magnification of the eyepiece was 15X giving 1/5 of the vial in view for counting at a total magnification of 22.5X. Larvae that hatched from the controls, 2.0 /u,g/ cm2 treatment (7 and 14 d, and continuous exposure), and 0.2 /xg/cm2 treatment (14 d, continuous exposure) were placed back onto chickens to evaluate feeding and molting capabilities of these larvae. The larvae were removed from the vials and placed directly on the chickens if possible. Any larvae remaining in the vial were allowed to find their way to the host by way of wooden applicator sticks and paper bridges from the bottom of the infestation chamber to the caged bird. One chicken was used for each dosage-exposure treatment, and each bird was isolated from the others. Engorged larvae were collected from each chicken and placed into individual 0.5-liter glass jars with fine mesh cloth covers. After all larvae had dropped, replicates of 25—30 engorged larvae from each treatment were transferred to 3.0-dram vials with screen tops and held in the environmental chamber as described previously. Up to 20 replicates of each dosage-exposure time were prepared, except for treatments where there were not enough engorged larvae. Replicates were scored for number of larvae molted, number of nymphs alive at 7 d after molt and the number of black and white fecal spots deposited after 30 d after molt, as described previously. Comparisons of means of selected variables were evaluated using the general linear models (GLM) and Tukey honestly significant difference (HSD) mean separation procedures (SAS Institute 1988) for tests of statistical differences (a = 0.05) for both engorged female and egg bioassays. Results and Discussion Engorged Female Bioassay. Table 1 summarizes results of exposing newly engorged A. americanum females to pyriproxyfen. There were no discernible differences in preoviposition time regardless of treatment. There were no statistical differences between treatments for the number of females ovipositing (Table 1), even at 16 /ng/cm2 where progressively fewer females oviposited with increasing

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TEEL ET AL.: PYRIPROXYFEN ON

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Table 1. Fecundity of lone star ticks exposed to pyriproxyfen as engorged females (re = 15) Treatment, Mg"

Exposure time''

Control 4 4 4 8 8 8 16 16 16

Control 7d 14 d Con 7d 14 d Con

7d 14 d Con

Mean % ovipositing^ (no.) ± SD 87(13) ± 93 (14) ± 100 (15)a 93(14) ± 100(15)a 93(14) ± 93 (14) ± 100(15)a 93(14) ± 80(12) ±

35.2a 25.8a 25.8a 25.8a 25.8a 25.8a 41.4a

Mean % hatch

No. unhatched egg masses with fecal spots

Mean % fecal spots in egg masses without hatch''

89 (n = 12)