By B. F. STONE*

INHERITANCE OF RESISTANCE TO ORGANOPHOSPHORUS ACARICIDES IN THE CATTLE TICK, BOOPHILUS MIOROPLUS By B. F. STONE* [Manuscript received August 29, 1967] Summary An organophosphorus·resistant strain of the cattle tick B. microplus from central Queensland was crossed with a susceptible reference strain, the ticks being confined in plastic mating boxes glued to the skins of cattle. The resistance of Fl backcross and F2 larvae was compared with that of larvae of the parent strains by exposing larvae to filter-paper packets impregnated with solutions of dioxathion, carbophenothion, and formothion in olive oil. Some comparisons were also made by similarly exposing engorged adult females to these compounds or by injecting them into engorged females. The relative resistance of hybrids from reciprocal crosses, and the segregation ratios obtained in backcross F2 and repeated backcross progenies were generally in satisfactory agreement with expectations for a single incompletely dominant autosomal gene. Segregation into phenotypes was clearest with formothion for which the degree of resistance in homozygotes was up to 5200 times and in heterozygotes up to 950 times. Homozygotes were up to 12 and 6 times resistant to carbophenothion and dioxathion respectively. 1. INTRODUCTION Resistance to organophosphorus compounds in the cattle tick Boophilus microplus was first reported from a property near Rockhampton in central Queens land by Shaw and Malcolm (1964), and Roulston et al. (1968) reported on the chemical control of a similar organophosphorus-resistant strain collected from an adjoining property where dioxathion had been in use since 1960. Control was satisfactory at first, but by early 1963 more frequent dippings were required, and later control was judged to be unsatisfactory. Studies on the inheritance of resistance to DDT and dieldrin in the cattle tick have been reported (Stone 1962a, 1962b) and this paper records similar work to determine the mode of inheritance of resistance to organo phosphorus compounds. II. MATERIALS AND METHODS The Ridgelands strain R (Roulston et al. 1968) was collected in October 1963 from the Rockhampton area in central Queensland, and after the strain was established at Yeerongpilly it was selected further with dioxathion in an attempt to produce homogeneity. Three resistant substrains were maintained: R g, to which selection pressure was applied usually at each generation; R e, selected almost continuously (every 2-3 days instead of at each generation) for dioxathion resistance; RR, obtained by selection of substrain R for low brai~ cholinesterase activity and the apparently associated homozygosity for resistance to organophosphorus compounds (Stone 1968). * Division of Entomology, CSIRO, Veterinary Parasitology Laboratory, Yeerongpilly, Qld.; present address: Department of Zoology, University of Western Ontario, London, Ontario, Canada. Aust. J. biol. Sci., 1968, 21, 309-19 310 B. F. STONE Selection pressure was normally applied by enclosing larvae for 16-24 hr in filter-paper packets impregnated with 7 f-Lgjcm 2 dioxathion. Occasionally selection was effected by injecting 5 f-Ll of a 0·23% dioxathion solution in olive oil (11· 5 f-Lgjg of tick) into engorged females, or by enclosing engorged females in filter-paper packets impregnated with dioxathion (219 f-Lgfcm 2 ), and breeding from the surviving females. Selected larvae produced by one of these three methods were used to infest steers. These larvae provided engorged nymphs which were allowed to moult in isolation to give the males and virgin females used for crossing as well as the engorged females which formed the basis of stock cultures of the selected resistant substrains for acaricide tests. The susceptible Yeerongpilly reference strain [previously referred to as strain Y (Stone 1962a, 1962b)] which had been cultured in acaricide-free isolation for 14 years was called strain S in accordance with the wide usage of "S" to denote susceptible strains. The crossing procedure was similar to that described by Stone (1962a) except that single-pair matings were often carried out as well as multiple-female (2-5 per mating box)x single-male matings, and mass matings (up to 10 of each sex per mating box). Mating boxes were of an improved screw-cap type cut from the neck of a polythene bottle, and organdie was used as a ventilating seal between the cap and base. The adhesive was a hot mixture of resin (colophony) and beeswax modified from a 4 : 1 to a 7 : 3 ratio. After they had detached, engorged females were removed from their mating boxes and placed for oviposition singly in vials and, unless stated otherwise, each resulting batch of larval progeny from a single female was tested separately. Most culturing and testing was carried out at 27°C and 80-90% R.H. but in one crossing experiment the incubation temperature was 35°C for some engorged females and egg batches. The following notation was used throughout to identify the progeny of crosses by their parentage, the female parent always being given first: F l : RS and SR; F2: F 2RS and F2SR; Backcross: RSjR, RjRS, SRjR, RjSR, RSjS, SjRS, SRjS, and SjSR. Larvae, usually 7-14 days old but on occasions up to 46 days old, and engorged females were tested in filter-paper packets impregnated with oil solutions of acaricides in the manner described by Stone and Haydock (1962). Packets were prepared from Whatman No. 541 filter papers after the application of the appropriate concentration of the acaricide made by serial dilution in a 1 : 2 olive oil-trichloroethylene mixture. Dosages were recorded in micrograms per square centimetre. * The olive oil used was sterilized injection grade, stabilized by the anti oxidant lonol (2,6-di-t-butyl p-cresol). A packet for the testing of larvae or unfed adults was prepared from one 11-cm paper but a packet for engorged females was made by clipping together two unfolded strips (7·5 by 9 em). In some tests engorged females were placed in an inverted 9-cm plastic Petri dish between two 9-cm filter papers each treated with 0·67 ml solution. The exposure time was 24 hr for larvae and unfed adults, and 24--48 hr for engorged females. Mortality was taken as the sum of the number of dead larvae and those so badly affected as to appear immobile when viewed at a magnification of 2-3 times. The responses of engorged females to the treatments were measured either quantitatively as "larva production responses" (Stone 1962a), or as "quantal responses" which were the per centage of females failing to lay viable eggs. For both these responses regression lines were fitted by eye (curved or straight lines; Hoskins 1963) and by maximum-likelihood calculations to the experimental points obtained by plotting response as probits against logarithm of dosage (Hoskins and Gordon 1956). These regression lines are referred to as ld-p lines. LD50 is the median lethal dose for larvae, and ED50 the median effective dose for engorged females. All relative resistances are calculated at LD50 or ED 50 values. Probit analyses of the quantal data were by means of a computer programme based on the method of Finney (1952). Where there was a natural response in controls the responses due to * A 1 % packet (Stone 1962a) is equivalent to 35 f-Lgjcm 2. INHERITANCE OF ACARICIDE RESISTANCE IN CATTLE TICKS 311 treatment were calculated by means of Abbott's formula which was also used to correct responses of resistant strains where there appeared to be a proportion of less resistant individuals in the sample. All ld-p lines in the figures were drawn with their true slopes but LDso values, relative resistance values, degrees of dominance, and fiducial limits were calculated using the common slope for the experiment. All expected responses for F2 and backcross progenies were calculated from the best-fitting ld-p lines for the parent strains and the FI, using the single-gene segregation ratios. It appeared from inspection of most quantitative data that there was a straight-line relationship between probit quantitative response and log dosage. Therefore ld-p lines of best fit were used but fiducial limits and significance levels are not stated because of the inapplicability of variances, weights, standard errors, and tests of significance calculated by methods of probit analysis designed for quantal data. The degree of dominance of resistance (D) was calculated as the excess of the heterozygote score over the mid-parental score expressed as a fraction of half the difference between the two parental scores, and is given by the formula cited by Falconer (1960). This may be expressed as follows: D = [X2-!(X1+Xa)]/!(X1-Xa) = (2X2-X1-Xa)/(X1-Xa), where Xl = log LDso of resistant homozygote, X2 = log LDso of hybrid, and Xa = log LDso of susceptible homozygote. Complete dominance is indicated when D = 1, incomplete dominance when 0 < D < 1, and no dominance when D = O. A negative value for degree of dominance signifies the corresponding positive degree of recessiveness. As resistance to dioxathion in strain R conferred cross-resistance to carbophenothion and formothion (Roulston et al. 1968), these three chemicals were used interchangeably in the following tests and the results interpreted accordingly. Dioxathion used was supplied as technical Delnav by William Cooper and Nephews (Australia) Pty. Ltd., carbophenothion was Trithion 95, and formothion was supplied by Chemicals (Queensland) Pty. Ltd. III. RESULTS (a) Fl Progeny of Reciprocal Crosses Results obtained by testing R e, RS, SR, and S larvae in formothion packets are compared in Figure 1. LD50 values (with 95% fiducial limits), slopes of the ld-p lines, and relative resistances (with 95% fiducial limits) are given in the following tabulation: LD50 Values Slope of ld-p Relative Strain (p.g/cm2) Lines Resistance Rc 63·5 (49·8 -81·3) 3·69 2380* (1740-3240) RS 10·2 (7'80-13'1) 3·88 380* ( 275- 521) SR 14·1 (10·9 -18·8) 6·63 528* ( 382- 731) S 0·0267 (0'0217-0'034) 3·95 1 * Significantly different from value for S strain at P < 0 .

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