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 . 05.

The slopes of the four ld-p lines do not differ significantly from one another (common slope = 4·12), and the lines for RS and SR larvae are similarly positioned. Although a comparison of the LDso values indicates SR larvae to be significantly more resistant than RS larvae, the tolerance distributions of the heterozygotes show a considerable overlap and the difference between the two sets of hybrids is slight compared with their differences from Rand S larvae. This test indicates that resistance to organo- 312 B. F. STONE

99-99 99-9 // 99 x -/ 95

90 ,...... ~ '-' >- :::;I- <{ 30 I-a: 0 I: If

/'/' x R LARVAE 0-' :Sl:::::E L :I::. SR LARVAE 0'01 ~ , , ~-~ ~-'$ ff--'-'--'-' -"-----'-- 0'00490 0'00980 0'0190 0·0385 0'0770 0-154 0-308 0-630 1·22 2-45 4'90 9'80 19'6 39'2 78'''' 157 FORMOTHION (fLG/CM2)

Fig. I.-Mortality of B. microplu8 larvae enclosed in formothion packets. Each percentage mortality is based on 110-260 larvae 7-lO days old.

99'99 6

,...... 99-9 ...... ,~ • w 99 '"Z 0 Il.. 95 Vl w a: 90 Z 0 80 i= 71) U 60 :> o RS ENGORGED FEMALES Cl 50 0 40 a: • SR ENGORGED FEMALES Il.. 30 <{ 20 >a: <{ 10 ...J

l' ,/"',,, 5'100 " 9'~~ 2t'Q I I 4~'O I 8;4-0 f?2 3~O 10·0 25'0 44-0 75'0 156 288 DIOXATHION (fLG/G)

Fig. 2.-Larva production response of engorged females of B. microplu8 to injection of dioxathion. Each percentage response is based on 50 ticks. INHERITANOE OF AOARICIDE RESISTANOE IN OATTLE TICKS 313 phosphorus compounds in larvae is incompletely dominant; the degrees of dominance calculated from RS and SR data are +0·528 and +0, 613 respectively. Furthermore there was no evidence of sex linkage or cytoplasmic inheritance and this was con­ firmed by injecting RS and SR engorged females with solutions of dioxathion in oil. The larva production responses for RS and SR engorged females are given in Figure 2. The slope of the ld-p lines (3· 08 and 3· 70 respectively), and ED 50 values (19, 6 and 21· 5 (Lg/g respectively) are not significantly different. Further samples of SR engorged females were tested in comparison with Rand S engorged females by enclosure in dioxathion packets and their larva production response measured (Fig. 3). ED 50 values, slopes of the Id-p lines, and relative resistances are listed below:

ED50 Values Slope of ld-p Relative Strain (p.g/cm2) Lines Resistance Rg* 181 3·18 5·68 SR 147 2·55 4·62 S 31·9 5·26 1·0 * Third generation of substrain Rg selected for one generation.

R type engorged females were slightly more resistant to dioxathion than were the SR engorged females. The degree of dominance calculated from the SR data was +0' 775, again indicating incomplete dominance. These results have detailed the findings for only one acaricide in each of the three types of test. Similar series of trials with the remaining two acaricides involving the progenies of 40 single-pair matings showed that similar results were obtained with all three acaricides. (b) Backcrosses R c, RS, SIRS, R/RS, and S larvae were tested in formothion packets and the results are shown in Figure 4. LD50 values, slopes of the ld-p lines (which do not differ significantly), and relative resistances are given in the following tabulations:

LD50 Values Slope of ld-p Relative Strain (p.g/cm2) Lines Resistance Rc 91·3 6·09 5170 RS 16·7 3·10 948 S 0·018 8·31 1

The degree of dominance calculated from RS data is +0·603. There is very good agreement between observed and expected SIRS mortalities particularly over the range of dosages for which 50% is the expected mortality. This finding provides strong evidence that, in larvae, resistance to organophosphorus compounds is due to a single gene. The R/RS mortalities likewise indicate segregation into two genotypes but the ratio is not as close to the expected 1 : 1 as in SIRS. Similar tests with dioxathion and carbophenothion using larvae of the F 1 X S backcross were less precise because of the lower orders of resistance, but led to the same conclusion. 314 B. F. STONE

1 1 ,,­ 99'99 ".- ",," .... " ,,"" ",,"" .,." "",'" Q9'9 "":.".",,,,,,,,;

• ;"" "",'" ,,-.. 99 "",-;."..""" '-" :,' *w V) Z 95 0 90 V)"- w a:. 80

Z 70 0 f= 60 u 50 ::l 40 0 Po. ENGORGED FEMALES 0 ,. a: 3l . S ENGORGED FEMALES 20 ,/ "- .- .- SR ENGORGED FEMALES « 10 >a:. « ...J

l'OIC" ! 27-3 54'6 109 219 .0438 875

DIOXATHION (fLG/CM2 ) Fig. 3.-Larva production response of engorged females of B. microplu8 after enclosure in dioxathion packets.

t t 99'99 6 6 6 6 o 1 .ll , / 99'9 .;/

99

95

90

,,-.. 80 '-'* 70 >­ 60 _._!---//// /. I-­ SO /-t-:l:-1~:~'- /~./ :::i ...... /;...... 1 « 40 ...... 30 ~ o 20 • : • """ c ~ RIR5 LARVAE • ./~ 10 ._. (OBS) 0 o RIRS LARVAE (EXP ~ ,/ ~ IX " I 0 RS LARVAF ) ~.~~o..,~ I'". SIRS LARVAE (OBS) ~ . ., "ciV ~-" ~;,"",""'" ",," """,,,",' ,/f/ / /. / ·'t / /" /, * -x' 0'00980 0'0190 0'0385 0'0770 0'154 0·308 0'6]0 1'22 2'.045 "'90 9'80 19-6• 39'2 78·" 157 FORMOTHION (fLG/CM2) Fig. 4.-Mortality of B. microplu8 larvae enclosed in formothion packets. Each percentage mortality is based on 120-240 larvae 7-11 days old. INHERITANCE OF ACARICIDE RESISTANCE IN CATTLE TICKS 315

The four possible types of engorged females of the FIX S backcross were combined and tested in dioxathion packets in comparison with R, S, and hybrid (RS combined with SR) engorged females, SR and RS larvae and engorged females having been shown previously to have similar responses to dioxathion. The ED50 values for R, S, and RS+SR were 156, 31· 9, and 118 t.Lg/cm2 respectively. Resistance to dioxathion relative to strain S for R and combined RS+SR was 4·9 and 3· 7 respectively. Larva production responses for these backcross females, and data for S larvae obtained previously, ·are shown in Table 1 and were used to calculate the expected backcross responses assuming a 1: 1 segregation ratio of hybrids to susceptibles. There is good agreement between observed and expected values at most of the 11 dosages and this provides strong evidence that resistance to organophosphorus compounds in engorged females, as in larvae, is due to a single gene. TABLE 1 LARVA PRODUCTION RESPONSE (P) AND MEAN WEIGHT OF LARVAE PER GRAM OF ENGORGED FEMALES (W) OF RESISTANT (R), HYBRID (RS+SR), SUSCEPTIBLE (S), AND COMBINED BACKCROSS STRAINS OF B. MICROPLUS ENCLOSED IN DIOXATHION PACKETS Each percentage response is based on 60 ticks Dioxathion Combined Backcross Dosage R Strain* (RS+SR) Hybrid S Strain Wobs. Pobs. P exp . (p.g/cm2) W(g) P(%) W(g) P(%) W(g) P(%) (g) (%) (%) 875 0·000 100 0·000 100 0·000 100 100 618 0·008 97·6 0·000 100 0·000 100 100 438 Q·001 99·7 0·006 98·4 0·000 100 99·9 309 0·000 100 0·011 96·9 0·007 97·8 99·2 219 0·073 78·5 0·024 93·2 0·000 100 0·032 90·5 95·2 155 0·192 41·5 0·087 75·0 0·082 75·3 85·8 109 0·287 15·7 0·181 48·2 0·003 98·8 0·088 73·4 71·4 77·4 0·301 11·5 0·308 11·9 0·150 55·5 57·3 54·6 0·330 5·5 0·042 85·9 0·179 46·1 44·3 38·5 0·269 19·0 29·0 27·3 0·220 25·9 0·294 11·4 13·8 19·6 0·303 8·7 4·35 Control 0·341 0·349 0·296 0·332 * Fourth generation, selected for two generations.

(c) F2 Progeny F2 RS engorged females, resulting from the interbreeding of RS adults were tested against carbophenothion in comparison with RR and S engorged females (Fig. 5). ED50 values (with 95% fiducial limits), slopes of the ld-p lines, and relative resistances (with 95% fiducial limits) are given below:

ED50 Values Slope of ld-p Relative Strain (p.g/cm2) Lines Resistance RR 815 (735-911) 7·02 11·9 (10,3-13,7) S 68·6 (62'3-75'5) 4·47 1

Slopes of the ld-p lines are not significantly different (common slope = 4·98). The inflexion in the F2 curve at about the 25% response level indicates segregation 316 B. F. STONE

99'99 l t/

99.J FIG. 5 *

99

95

~""' 90 '-' w 80 VI Z 70 0 60 "- Vl 50 w " ~« C" " " ~:::l (Y 5 ~~/ )( RR ENGORGED FEMALES

.6 F2 RS ENGORGED FEMALES (OBS.)

/ 0 S ENGORGED FEMALES I 0'1 I I I 0'01 I ,I 27'7 39-2 55-4 78-4 III 157 221 313 443 627 887 1254 1774 2508 CARBOPHENOTHION (fLG/CM 2)

99-99 6 /" 1 1 .;.; .;.; 99-9 FIG. 6 / // .; 99 ,., ,., 95

90

""' 80 ~ / "/ '-'~.>- 6070 / / ,4c """" ,,'' ~~. " ,_ .~, C'O" ,/ / / g" E r ~ ,~ccmo,' ~• / ." ,~'''' ,,'/ / 10 . " """ ,/ / ;.w" "cc"" / / ;."" "" '''00: '" '" ,0"'., // / / / ; "'''' """ "'" // / / ~ /' o /

0'1 / // // / // // 0·01 , / / ~/

0'00720 0'0140 0'0390 0-0780 0-155 0'610 2'45 4\0 9-80 19'6 39-2 FORMOTHION (fLG/CM 2)

[For legends to Figures 5 and 6, see opposite page.] INHERITANCE OF ACARICIDE RESISTANCE IN CATTLE TICKS 317 into resistant and susceptible phenotypes in approximately the 3: 1 ratio expected if resistance is considered to be due to a single gene. Similar tests with dioxathion against F 2 1arvae led to the same conclusion but were less precise because of the lower level of resistance to this acaricide.

(d) Repeatedly Selected Backcrosses Larval progeny of S adults mated with RSjS adults which had been selected for dioxathion resistance with a dosage which discriminated against susceptibles were tested in packets at serial concentrations of formothion, in comparison with R, RS, and S larvae. The results given in Figure 6, when compared with those in Figure 4, show that double backcrossing after elimination of susceptibles does not alter the 1 : 1 ratio of resistants to susceptibles, and that the order of resistance of the resistant fraction in the second backcrosses is not less than that in the first backcrosses. This finding provides further evidence of the monofactorial nature of resistance to organophosphorus compounds. The high level of resistance to formothion permitted the confident use of discriminating dosage tests to determine the proportion of resistant and susceptible larvae in a further 10 double­ backcross progenies. The results of such packet tests show that all but two of these progenies gave mortalities not significantly different from the expected 50%, and the pooled mortalities also did not differ significantly from 50%. These results confirm the 1 : 1 ratio of resistant to susceptible progeny in these repeated backcrosses.

IV. DISCUSSION The backcross tests with formothion provided the strongest evidence for monofactorial inheritance of resistance to organophosphorus compounds. The clear-cut nature of these results was due principally to much wider separation, with no overlap between hybrid and susceptible distributions of tolerance, for formothion than for carbophenothion or for dioxathion, the maximum relative resistance factors for hybrid larvae being 950, 23, and 4·6 respectively (Stone, unpublished data). It was considered legitimate to use formothion as it is widely accepted that in a strain of arthropods where selection with one toxicant produces resistance to that toxicant and cross-resistance to a number of other toxicants which have not been used to select the strain, the cross-resistance is due to the same resistance mechanism (Milani 1963). Thus, there seems to be little doubt that the resistance of strain R of B. microplu8 to the three organophosphorus compounds is due to a single autosomal gene incompletely dominant with respect to its susceptible allele. In a recent review Milani (1963) concluded that 23 out of 27 instances of insecti­ cide resistance subjected to genetic analysis involved monofactorial inheritance. More specifically, resistance to organophosphorus compounds was noted to be

Fig. 5.-Quantal response of engorged females of B. microplu8 after exposure between carbo­ phenothion-impregnated filter papers in plastic Petri dishes. Each percentage response is based on 40 ticks. Fig. 6.-Mortality of B. micropluslarvae enclosed in formothion packets. Each mortality is based on 120-190 larvae. 318 B. F. STONE monofactorial in six out of seven examples cited, and in each case resistance was dominant or nearly so. Since Milani's review there do not appear to have been any papers which materially change this situation except that Dittrich (1963) reported that resistance to methyl demeton in a strain of Tetranychus urticae was caused by multiple dominant semilethals. One of the complicating factors in this investigation was the heterogeneity of the original strain R as this precluded the confident use of mass mating. However, in a few early tests where mass mating was used, each engorged female was isolated and her progeny tested separately. Although there is little doubt that B. microplus males are capable of repeated copulations with different females it is not known whether they will attempt to copulate with or be accepted by a previously mated female. Later either single-pair matings, or multiple-female x single-male matings were used and their use removed any slight doubt as to the reliability of crossing tests. It is interesting to compare the rates of development of the three types of acaricide resistance recorded in B. microplus. Dieldrin resistance, which is com­ pletely dominant (Stone 1962a), developed extremely rapidly (Stone and Meyers 1957 - 7 months, 3 sprayings; Roulston 1964 - 4 months, 4 dippings) whereas DDT resistance, which is incompletely recessive (Stone 1962a), was not detected until about 8 years after DDT was first used (Stone and Webber 1960). Dioxathion resistance, now shown to be incompletely dominant, developed at a somewhat inter­ mediate rate in strain R after approximately 3 years use of dioxathion (L. G. Webber, personal communication) so it is tempting to suggest that there is a correla­ tion between rate of development of resistance and degree of dominance. However, the rate of development of resistance must also be influenced by the frequency of resistant genes in the original populations and the intensity of selection factors for which information is not available.

V. ACKNOWLEDGMENTS Grateful acknowledgment is made of the help of Mr. G. A. McIntyre, Division of Mathematical Statistics, CSIRO, Canberra, who wrote the computer programmes for probit analysis and processed much of the data. Thanks are due to Dr. R. H. Wharton and Mr. R. W. Kerr, Division of Entomology, CSIRO, to Dr. A. W. A. Brown, Department of Zoology, University of Western Ontario, and to other colleagues for encouragement, advice, and help with the preparation of the manu­ script. The author is also indebted to Mr. L. G. Webber, Imperial Chemical Industries of Australia and New Zealand Ltd., who supplied the organophosphorus­ resistant strain R, and to Misses W. H. Ward and N. J. Youlton for technical assistance. VI. REFERENCES

DITTRICH, v. (1963).-Genetics and dynamics of organophosphate resistance in strains of Tetranychus urticae. Adv. Acar. 1,238-47. FALCONER, D. S. (1960).-"Introduction to Quantitative Genetics." (Oliver and Boyd: Edinburgh and London.) FINNEY, D. J. (1952).-"Probit Analysis." (Cambridge Univ. Press.) HOSKINS, W. M. (1963).-Resistance to insecticides. Int. Rev. trap. Med. 2, 119-74. INHERITANCE OF ACARICIDE RESISTANCE IN CATTLE TICKS 319

HOSKINS, W. M., and GORDON, H. T. (1956}.-Arthropod resistance to chemicals. A. Rev. Ent. 1, 89-122. MILANI, R. (1963}.-Genetical aspects of insecticide resistance. Bull. Wid Hlth Org. No. 29. Suppl. on Vector Control. pp. 77-87. ROULSTON, W. J. (1964}.-A study of the development of dieldrin resistance in relation to acaricide pressure in a population of Boophilu8 microplu8. AU8t. J. agric. Res. 15,490-510. ROULSTON, W. J., STONE, B. F., WILSON, J. T., and WHITE, L. 1. (1968}.-Chemical control of an organophosphorus- and carbamate-resistant strain of cattle tick (Boophilus microplus) from the Rockhampton area of Queensland. Bull. ent. Res. (In press.) SHAW, R. D., and MALCOLM, H. A. (1964}.-Resistance of Boophilus microplus to organophosphorus insecticides. Vet. Rec. 76, 210-11. STONE, B. F. (1962a}.-The inheritance of DDT resistance in the cattle tick Boophilus microplus. Aust. J. agric. Re8. 13, 984-1007. STONE, B. F. (1962b}.-The inheritance of dieldrin resistance in the cattle tick Boophilus microplus. Aust. J. agric. Res. 13, 1008-22. STONE, B. F. (1968}.-Brain cholinesterase activity and its inheritance in organophosphorus­ resistant and susceptible cattle ticks, Boophilus microplus. Aust. J. bioI. Sci. 21, 321-30. STONE, B. F., and HAYDOCK, K. P. (1962}.-A method for measuring the acaricide susceptibility of the cattle tick Boophilus microplus (Can.) Bull. ent. Re8. 53, 563-78. STONE, B. F., and MEYERS, R. A. J. (1957}.-Dieldrin-resistant cattle ticks, Boophilus microplus (Canestrini), in Queensland. Aust. J. agric. Res. 8, 312-17. STONE, B. F., and WEBBER, L. G. (1960}.-Cattle ticks, Boophilus microplus resistant to DDT, BHC, and dieldrin. Aust. J. agric. Res. 11, 105-19.