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Effects of circadian rhythms and other bioassay factors on cat flea (Pulicidae: Siphonaptera) susceptibility to insecticides.

2000; Kansas (Central States) Entomological Society; Volume: 73; Issue: 1 Linguagem: Inglês

1937-2353

Robert L. Bossard, Alberto B. Broce, Michael W. Dryden,

Animal Ecology and Behavior Studies

Variability in insecticide susceptibilities during nylon-disk bioassays of adult cat fleas (Ctenocephalides felis) due to the time of day the bioassay was conducted, illumi nation, density of insects, sex ratio, and C02 exposure was investigated. Also examined were illumination effects on flea circadian rhythms, as recorded by time-lapse video under 4 light ing conditions (12:12 L:D, 12:12 L:D with photophase at night, continuous light, or contin uous red light simulating darkness), and respiration in bioassay tubes. The time of day when the bioassay was started (00:30, 7:00, 12:30, 18:30), dark or light exposure, density (5, 10, 30 fleas/bioassay tube), and sex of the flea had no effect on mortal ity. After 24 hr, fleas that had been immobilized with C02 during sexing were more suscep tible than unsexed fleas not exposed to C02. At 4 and 16 hr, there was no difference. Unsexed fleas immobilized with C02 had intermediate susceptibility. Fleas showed 24-hr circadian rhythms. When under 12:12 L:D and continuous red light, peak activity was at 21:00 while respiration peaked at 18:00. Activity shifted 6 hr earlier un der 12:12 L:D reverse photoperiod. Under continuous light or dark, endogenous rhythms oc curred. Respiration of fleas in darkness was usually lower than in light. Though activity of fleas varied, insecticide susceptibility was not affected by these conditions. Other factors must contribute to high variability in cat flea susceptibility. Detection of insecticide resistance is difficult with high variation in susceptibility of insects during bioassay (National Research Council, 1986; Robertson et al., 1995). This is particularly true for cat fleas Ctenocephalides felis (Bouche) (Bossard et al., 1998) when the LC50 of a strain can vary up to 7-fold (Moyses, 1995). This variabil ity may be created, in part, due to a varying environment that affects insect activity and thus potentially changes the insects' contact with toxic residues (Shepard, 1960). Insecticide susceptibility of fleas is affected by temperature (Busvine and Lien, 1961) and age (El-Gazzar et al., 1988). Other factors that often vary between groups of fleas in bioassay tubes include time of day the bioassay is started, illumination, density of test insects, sex ratio, and use of C02 for anesthesia. Changes in the intensity of illumination can influence flea activity (Busvine and Lien, 1961; Dryden and Broce, 1993). A density of 10 fleas per 25 x 150 mm test tube is recommended (WHO, 1981) but during bioassays, this number may vary. In creased density during testing often affects insect physiology (Shepard, 1960). Os brink and Rust (1984) found single females in host-feeding cells had higher fecun dity than females placed 4 or more per cell. Sex ratio of flea samples varies as females emerge before adults (Dryden and Smith, 1994); so susceptibility of groups of fleas may vary also. Male insects are usually more susceptible to insecticides than females (Shepard, 1960). Dryden and Gaafar (1991) found male fleas less susceptible to systemic insecticides than females, 1 Current address: Sch. Biol. Sci. 4236, Washington State Univ., Pullman, Washington 99164. 2 To whom correspondence should be addressed. 3 Dept. of Diagnostic Medicine and Pathology, Kansas State Univ., Manhattan, Kansas 66506. In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. The facilities are fully accredited by the American Association of Laboratory Animal Care. This content downloaded from 207.46.13.163 on Fri, 08 Jul 2016 06:00:23 UTC All use subject to http://about.jstor.org/terms 22 JOURNAL OF THE KANSAS ENTOMOLOGICAL SOCIETY though Busvine and Lien (1961) found no significant differences of LC50's between sexes using contact insecticides in WHO bioassays of fed fleas. C02 immobilization, which is often used to make fleas easier to handle, increases insecticide susceptibil ity of cat fleas (El-Gazzar et al., 1988). The current study investigated whether insecticide susceptibility of cat fleas is af fected by the time of day the bioassay was started, illumination and density of fleas in bioassay test tubes, and C02 immobilization for separation by sex. Also investi gated were the effects of illumination on circadian activity rhythms and respiration as an indicator of the activity of cat fleas. Materials and Methods cat fleas: Fleas in this study were of a strain collected from naturally infested dogs and cats at an animal shelter in Manhattan, KS, and that had been in colony since July 1990. They were maintained in darkness (Dryden, 1989) and during bioas say were adults aged 0-3 days. Voucher specimens were deposited in the Entomol ogy Research Collection, Kansas State University, Manhattan, KS (Lot# 073). bioassays: Chlorpyrifos (Dursban XP, 99%; Whitmire Research Laboratories, St. Louis, MO) was weighed into 50 ml volumetric flasks and dissolved in acetone. Fifty jlxI of the solution was applied at 10 mg (AI)/m2 (except for the time of day experi ment when 50 mg (AI)/m2 was used) to a horizontal, 415 mm2 circular Nylon 6,6 disk (Cerex? Spunbonded Nylon?Type 23; Cerex Advanced Fabrics, L.P., Can tonment, FL) equal in area to the bottom of the flat-bottom glass test tube (25 x 150 mm) (Bossard, 1997). Disks dried on wire racks for at least 2 hr before testing. For circadian activity and respiration measurements, untreated strips (15 x 50 mm) of cellulose filter-paper (Whatman #1) were placed in these test tubes, similar to the WHO flea bioassay (WHO, 1981). Ten fleas were placed in each tube, except where noted for the density experiment. Tubes were capped with Parafilm?, which was punctured with needles, and placed upright at room conditions of ca. 20?C and ca. 40% RH. After treatment, fleas that were not moving or not oriented upright were recorded as dead. Percent mortalities were calculated, arcsin-transformed and analyzed by analysis of variance. Means were separated by pairwise f-tests and Fisher's least-significant-difference test at P = 0.05 (SAS Institute, 1988). time of day: Bioassays were started at 0:30, 07:00, 12:30, and 18:30 with insec ticide-treated (n = 6) and untreated (n = 2) nylon. Mortality was checked after 4 and 11 hr. illumination: Tubes with fleas were placed either under a box for darkness (ex cept when checking mortality during which fleas were exposed to light for a few minutes) or under a fluorescent light. Mortality on insecticide-treated (n = 6) and un treated (n = 2) nylon was counted at 4 and 24 hr. flea density: Densities of 5, 10, and 30 fleas per tube were assayed on insecti cide-treated (n = 5) or untreated (n = 2) nylon. Mortality was recorded after 4 and 24 hr. co2 and sexing: Fleas were exposed to enough C02 for 15 min to immobilize and separate them by sex. In addition to 'females with C02' and 'males with C02' groups, a group of C02-immobilized fleas were not segregated by sex. Fleas in a fourth group were neither sexed nor given C02. All groups were placed on insecticide-treated (n = 4) or untreated (n = 3) nylon. Mortality in tubes was monitored at 4, 16, and 24 hr. This content downloaded from 207.46.13.163 on Fri, 08 Jul 2016 06:00:23 UTC All use subject to http://about.jstor.org/terms VOLUME 73, ISSUE 1 23 Table 1. Mean percent mortality ? SEM of C. felis on nylon disks treated with chlorpyrifos 50 mg (AI)/m2, started at 4 times of day (00:30, 07:00, 12:30, 18:30).