- Email: [email protected]
Juvenile hormone analogs: Toxicity and cross-resistance in the housefly
PI’:‘;‘PI~‘II)~~
BIOCHEMISTRY
Juvenile
ANLI
Hormone
F.
3, 131-136
PHYSIOLOGY
ff.
liewived
Analogs: in the
PLAPP,
Novetnber
JR.,
Toxicity Housefly
AND
‘27, 1!)7:!
s.
and
BRADLEIGH
; accaepted
Cross-Resistance
l VINSON
March
LO, 197~1
The toxicity of several juvenile hormone analogs (JHAs) to susceptible and inaectitide-resistant housefly (MUSCU tlomcsticu L.) strains was det,ermined by an assay procsedure in which larvae were exposed to residues of JHAs in glass vials. All JHAs tested were toxic and the most active compound, isopropyl 11-methoxy-if, 7, ll-t,rimet,hylododeca-2, 4-dienoate, was 100 times as toxic to the susceptible Orlando Ilegular strain as methyl parat,hion and 600 times as toxic as DDT. A .i- to SO-fold tolerance to the different JHAs was present in an insecticide resistant &rain in which resistance is associated with a high level of NADPH-dependent microsomal oxidase activity controlled by a gene(s) on chromosome II. Cross-resistance was less marked in a strain wit,h a chromosome V high oxidase gene and absent in strains with ot,her resistance mechanisms. The data indicate that cross-resistance to JHAs in insects may occur in certain strains with high levels of &dative detoxifying activity. Even so, the most act.ive JHA was far more toxic t.u both susceptible a,nd resistant strains than methyl parathion or l)l)T. INTRODUCTION
Insect) juvenile hormones (JH) and their analogs (JHAs) have rccciwd a great, deal of attcwt,ion in the last scvcral years as possihlc inswt control agents. Uy disrupt.ing mc+amorphic dwrlopmcbnt, both JH and its analogs show varying dcgrws of insecticidal propcrtks. Thwcl propcrtiw arc so diffcrclnt from those of presently used synthetic organic insrct’icidcs that Williams (1) has wfcrwd to JH as a “third-genwation” Among t,hc advantagw over inwcticidr~. synthct,ic insccticidw arc wlectivity, high and thr fact that it would bc activity, difficult for an ins& to bwomc wsistant to its own hornlow. The effects of JH on diptcra wcrc first wported for AecZes aegypti 1,. (2, 3). 1 Approved 10’%7, Texas
copyright ~11 rights
for publication as Technical Art isle Agricldt.ln+al Experiment St,ation.
0 1973 by .&cademic Press. Inc. of reproduction in any form resxved.
Srivastava and Gilbrirt (4) showed t’hat JH affected pupal formation of Xarcopha!qa bdlata Parker and that th(t affrckd insects failed t,o cmc~rgc. Similar rcwlt,s \v(‘L’(’ wportcd for Roir~ft.~~stalcitrarrs (I,.) (5, 61 and for Nusca dwmestica (I,.) (7,. Sinw Williams’ statc~meiit, (1) t#hat t,hfb dwclopnwnt of rwist~ancc b.v an insect to its own hormotw n-ould bc difficult, th(t widespread assumption has been that inswts would not bccomc resistant to JH or JHA compounds. Howcvcr, since diffcrcwt. insect fatnilics vary in their response to particular JHs or JHAs (X-lo), it is possihlc t’hat diffcrenws may also occur wit,hin a spcciw. Variations in such paramctcw as penetration, metabolism, excr&~n, and/or tissue sensitivity occur bekecn inscct,icidt:susceptiblr and rwistant stra.ins (11) and thwt arc’ no good wasons why such diffw-
132
PLAPP
AND
ences should not affect hormone sensitivity as well. Several reports of tolerance to JHAs have appeared. A &fold tolerance to a JHA is present in a multiresistant strain of the tobacco budworm, Heliothis virescens F. (12), and a similar level of tolerance to a JHA occurs in a multiresistant strain of the red flour beetle, Tribotium castaneum (Herbst) (13). A tolerance to JHAs in insecticide-resistant houscflirs has bcrn described in a preliminary report from our laboratory (14) and in work by Cerf and Georghiou (15). In the latter case, tolerances of up to 40-fold were found at the LD,, level. To determine the nature of the relationship between insecticide resistance and resistance to JHAs, a number of housefly strains, with known mechanisms of resistance to insecticides, were exposed to several JHAs. By comparing the responses of these strains we hoped to determine which resistance mechanism(s) was responsible for JHA tolerance in houseflies. MATERIALS
AND
METHODS
The larvae of test housefly strains were reared on standard CSMA media. Adults were fed a 1: 1 mixture of sugar and powdered skim milk ad lib. A description of the strains used in thflse cbxpcriments follows : Orlando Regular-Insccticido-suscept,ible wild-type strain. stw; bwb; ocra-Susceptible strain homozygous for the visible recessive mutants stubby w&g, brozull body, and ocra eye, cont,rolled by genes on chromosomes II, III, and V, respectively (16). R-Fc-Resistant to DDT, carbamates, and organophosphates. Major resistance mechanism is a chromosomr V gene for high microsomal oxidase (16). R-Baygon; bwb; ocra-Resistant to carbamates, organophosphates, DDT, and cyclodienes. Major resistance genes arc chromosome II factors for high oxidase
VINSON
activity and DDT dehydrochlorinase and a chromosome IV factor for resistance to dieldrin and related compounds. The mutants b,wun body and ocra eye were introduced into the strain to eliminate resistanctx factors associated with chromosomes III and V, respectively (16). Other insecticide-resistant strains tested included Orlando DDT which has chromosome II and III genes for resistance to DDT (17), DDT-R; dov with a chromosome II gene for DDT resistance (17), kdr-0; ocra;stw with a chromosome III gene conferring resistance to DDT and pyrcthrins (17), Dld-R;cyw with a chromosome IV gene for dieldrin rcsist,ance (18), and R-Para;clw with a chromosome V altrrcd ali-&erase gene for parat#hion rrsistanrc (19, 20). Levels of oxidative detoxifying activit.y for the various resistant strains have been established previously. As measured by aldrin epoxidation in vitro, R-Baygon ;bwb ; ocra and R-F, have 2.4 and 2.0 times the activity of the st.w ;bwb ;oc.ra strain (16). In comparison with the Orlando Regular strain, aldrin epoxidasc in R-F, is four times higher than normal (21). Oxidasc activity in Orlando DDT, DDT-R;dov, kdr-0; ocra;stw, Dld-R;cyw and R-Para: clw is not significantly different from that of Orlando Regular flies (21). The JH analogs used in the tests arch described by their USDA-assigned ENT numbers and by formula. They are as follows : ENT 70033, trans-4- (6,7epoxy-3,7dimct’hyl-%octanyl)oxy-1,2-(methylenedioxybenzcne) ; ENT 70221,6,7-epoxygcranyl (p-ethyl) phenyl ether; ENT 70459, ct’hyl 3,7,1 I-trimethyldodcca-2,4-dienoate ; and ENT 70460, isopropyl 1 I-methoxy-3,7,1 ltrimethyldodeca-2,4-dienoats. Insecticides tested were DDT (technical grade) and methyl parathion (90%). EKT Nos. 70033, 70459, and 70460 were obtained from the Zoecon Corp., Palo Alto, CA. ENT No 10221 was obtained from the Stauffcr Chemical Co., Mountain View, CA. The
JUVENILE
HORMONtX
TABLE Toxicity
Data
AND
1
on the Response of Four Housejly Strains Hormone Analogs and Insecticides LCSos
Housefly
Orlando
strain
Regular
Valuea
I&l R value
stw ; bwb ; ocra
LCSO
R-Fc
LC60
R value
R-Baygon -.,~
; bwb ; ocra
R value LCjO R value
Methyl parathion 1.9a 1 3.5b 1.8 3.3b 1.7 92.6c 49
133
HOUSEFLIES
(rg/vial)
DI>T
12.6a 1 lX.Oa 1.5 154.9b 12 > 1,oooc >80
to Some Juvenile
and resistance
values
RNT 70033
ENT 70221
ENT 70459
ENT 70460
17.5a 1 81.3b 4.6 90.4b 5.1 422.5~ 24
35.3a 1 58.4a 1.6 122.4b 3.4 186.2~ 5.3
5.la I 10.9b 2.1 lO.Ob 2 34.7c 6.8
0.02a 1 O.llb - 0.r) o.lT,b 7.5 0.63~ 31
-
a LC50 values based on emergence of flies as adults. Resistance several strains to that of Orlando Regular flies. LCSo values followed different at the 95% confidence level.
DDT sample was purchased from Nutritional Biochemical Corp., and methyl parathion was obtained from American Cyanamid Corp., Princeton, XJ. The assays were performed by exposing housefly larvae to films of the test chemicals in glass vials. Mature, third (last)-instar fly larvae were collected from the rearing media 4 days after eggs were laid. Larvae of this age have usually stopped feeding as evidenced by clearing of the gut. Pupation normally occurred 24-45 hr after the tests wvcre initiated. Test solutions of all chemicals were prepared at 170 (w/v) or lower concentrations in acetone. Required amounts in 0.1 ml or less solvent were pipctt’ed with microsyringes in to glass vials of the type used for liquid scintillation counting. With additional acetone, the final volume was made up to 0.3 ml. The vials were then placed on t,hcir sides and rotated manually until the solvent was evaporated. Control vials were treated with acetone only. Twent’y larvae were placed in each vial and the top was covered with chiffon cloth and fastened with a rubber band. The vials were placed in an incubator at 27°C with a 14: 10 light: dark cycle. After 10 days t’he vials were removed and the number
values are the rat.ios of response of the by the same letter are not significantly
of flies which had successfully emerged was determined. Failure of adults to complete emergence normally was t’he criterion of assay. The results were analyzed by an IBM 360 computer program to determint IDSa values for the several chemicals in micrograms chemical/vial. In some experiments we determined the sex of emerging flies. However, since no differences relating sex to susceptibility were observed, the practice was discontinued. We also performed preliminary experiments to determine if exposure of flies to sublethal dosesof JHA resulted in a reduction in reproductive potential. Again, results were negative and the tests were discontinued. RESULTS
Data from the various bioassays are summarized in Tables 1 and 2. All results are based on a minimum of t’hree assays performed with different populations of flies and different solutions of the test materials. Table 1 presents the responses of two susceptible and two insecticide-resistant strains to all test, chemicals. The resistant strains, It-Fc and R-Baygon ;bwb ;ocra, possess resistance factors involving high levels of microsomal oxidase activity. The
134
PLAIT
TABLR Toxicity
Data
AND
2
on the Rcnponuc of Sir Strains to ENT XL@0
Housefly strain
LC:5,, (pgjvial )‘L
Orlando Regular Orlando lII>T DDT-13, ; dov kdr-0 ; ocra ; stw Did-ll; cyw It-Parathion ; clw
0.02a 0.03b O.&k 0.09c 0.01x 0.Old
Horwcfly
I(esistance value 1
2.5 4 4.5
0.5 0.5
u LCS,, values followed by the same letter are not significant)ly different at the %@,;,confidencelevel.
discussion that follows is based on the response of each strain t,o t)hc test compounds. All test materials were highly toxic to Orlando Regular house flies. Met,hyl parathion and DDT, both highly toxic insecticides, produced 50% mortalit’y at doses of approximately 2 and 12 pg/vial, respectively. Two of t.hc JHAs, ENT 70032 and ENT 70321, were less toxic than DDT by factors of 1.5- and 3-fold, rc>spcctively. ENT 70459 was intermediate in toxicity between DDT and methyl parathion. ENT 70460 was t,hc most toxic JHA tchsted. This matrrial was 100 t’imes as toxic as methyl parat,hion and 600 times as toxic as DDT. The response of the stw ;b\vb ;ocra strain followed a similar but not identical pattern t,o that of Orlando Regular flies. The major diffrrcncc \vas t,hat with all compounds tested, st,w ;bwb ;ocra larvao required 1.55.5 times as high a dose as those of Orlando Regular larvae for 50% mortalit,y. Wit.h all materials except DDT and ENT 70321 the difference in response between Orlando Regular and stw ;bwb ;ocra strains was statistically significant. This was surprising since previous tests with conventional insecticides (16) indicated that stw;bwb; ocra adults are nearly identical in response to Orlando Regular adults. Strain R-Fc, with high oxidasc activity rrsistan(*(b
to insc&c:idc~s
controlkd
by
a
gene(s) on chromosome 1’ responded simi-
VINSON
larly to t.hc stw;bwb;ocra strain to all compounds except) DDT and ENT 70221. With DDT, a lo-fold rcsistanct~ level \vas present. With ENT 70221, the LCso for R-Fc was t,wicc as great as that for stw ; bwb;ocra flies and nearly four times that of Orlando Regular flies. The R-Baygon :bwb ;ocra strain consistently showed the highcast lcvt~l of resistance t,o all chemicals tested. With methyl parathion and DDT, rcbsistancc levels were 4%fold and greater than 80-fold, respectively. Wit,h the JHAs, the dose Ievrls required for 50% mort,alitg n-erc 5- to 30-fold greater than for Orlando Regular flies. Resistance was highrst with EN’1 70321 and ENT 70460. In both cast’s th(l LCsO valurs wclre 20- to 80-times greater t,han for Orlando Regular flies and fivchtimes great,er than for stw;bwb ;ocra flks. In all cases, LCsas were significantly higher for this st’rain than for all othcbrs. Additional tests were’ condu&d with ENT 70460 on several othcbr insc&cido resistant strains. The r(ksults (Table 2) indicate this JHA is highly act#ive against all
test
fly
strains.
Tht:
compound
\vas
more toxic to dieldrin- and parathionresist,ant strains than to Orlando Regular flks. The three DDT-resistsant strains w(bre only slightly less susceptible. It is worth not,ing that none of the strains list>ed in Table 2 possess high levc~ls of micrtrsomal oxidasc act,ivity as a component of thrir rcsistanre spectra. DISCUSSION
The data show that some JHAs are highly toxic to houseflies treated as larvae and that the most active compound, ENT 70460, is far more toxic than standard insccticidcs. The dat,a also confirm previous findings (12-15) of c~ross-rc~sist’ancc, to JHAs in insecticide-resistant st#rains. Our result’s show that cross-resistance is highest in the R-Baygon ;bwb ;ocra strain which has chromosomfl II g;c~nc~s for high microsomal oxidascb acativity and DDT dehydrochlorinase as well as a chromosome
.TI:VENIT,E:
HORMONES
IV gene for cyclodiene resistance. Since other strains with the DDT dehydrochlorinase- and cyclodicnc-resistance genes were susceptible to the test chemicals, it is likely t#hat# the chromosome II high oxidaso gcnr is responsible for rcsistancc~. Ilouseflies possessing this factor havcl previously been shown to mctabolizca man) irisc&c*idcn, i.cb., aldrin, allrthrin, caarbaniatrs, and organophosphat,c~s at grcbatcr rates than suscrptibh: flies (16). The reason for the relatively higher rclsistance to JHAs of R-Baygon;bwb ;ocra as compared with R-Fc is not’ known. Both strains have high oxidase activit,y as measured with aldrin epoxidasc>. HOWWW. nit,h other insccticidc chemical substratcls, differences exist brtwccn tht> st#rains. For rhxamplc, It-Baygon ;bwb ;ocra Aics metabolize allethrin brttcr than R-Fc flies while with DDT thr convcrsc is true (10). Thus, qualitative diff (rcnces exist, bctwecln th(k strains and th(ly art’ apparcnt)ly rcflcrtc~d in diffcrcnces in rc’sponscb to JH.4s as well. Published data (22) on th(l role of hydroxylation and cipoxidatjion in hormonc~ mc%abolism support the hypot’hesis that microsomal oxidases arc important, in JHA mc+abolism. Th(rc is at present, no adequat,(x biochemical rvidcncc that this microsomal oxidasck systc>ni is involvf)d in rrsistancc. lIowev(r, thcb cxar1ic.r report, (23) of JH activit,y of microsomal oxidascl inhibit,& synrrgists and tht> recent finding (24) that JlIAs arc microsomal oxidasc inducers further implicate the system as of prime importance in JHA toxicity. The occurrence of cross-resistance to JHAs in the housefly strains resistant’ to insc&cidcs suggests t)hat c’xposure to prcscnt~ly used toxicants may lrssc~l the potential of JHAs as control agmlts. F’urthrrmort>, this c~xperimr~ntally induccbd cross-rcsist,ance to JHAs implies that even highc~r 1~~~1s of rcsistancc may develop wl1c.n ins& populations arc exposed to horuion(~s in cont,rol programs. If this is tru(a, th(>n the use of JH and JHA materials may not. be the solution to resistance
AND
135
HOVSEFLIES
problems originally hoped for. However, it is worth noting that even with resistance bating prctsclnt, the most’ active JHA is morcx toxic to housefly larvae of bot)h susceptible and resistant strains than either DDT or mc+hyl paratjhion. ACKNOWLEDGlIENTS
We thank Ms. E. A. Butler for technical assistance during the course of these experiments. 1)r. J. (+. Benskin, formerly of this laboratory, participated in a number of preliminary experiments which led to this publication. REFERENCES
1. C.
2.
:<.
4.
5.
6.
7.
6.
9. 10.
11.
12.
hl. Williams, Third-generation pesticides. Insect hormones and their analogs could provide highly selective insecticides, Sri. Amrr. 117, 13 (1969). A. Spielman and C. M. Williams, Lethal effects of synthetic juvenile hormone on larvae of the yellow fever mosquito, A&s aegypti, Scimcr 154, 1043 (1966). A. Spielman and V. Skaff, Inhibition of metamorphosis and of ecdysis in mosquitoes, J. Znwrt Phgsiol. 13, 1087 (1967). V. S. Srivastava and L. I. (Gilbert, Juvenile hormone : Effects on a higher dipteran, Scicncc~ 161, 61 (196X). J. E. Wright, Hormones for control of livestock arthropods. Development of an assay to select candidate compounds with juvenile hormone activity in the stable fly, J. Econ. Entornol. 63, x7x (1970). J. Ji:. Wright. and G. h:. Spates, Biological evaluation of juvenile hormone rompounds against pupae of the stable fly, J. Agr. Food Chew. 19, 289 (1971). A. Herzog and B. E. Monroe, Effect of synt.hetic juvenile hormone and citric acid on housefly pupae, ,II usca domrstica I,., Camp. RiochwL. Phz/siol. 41, 481 (19723. J. J. Glenn and IVI. 13eroza, “InsecBt Juvenile Hormones : Chemistry and Action,” 341 pp., Academic Press, New York, 1972. 6. Slama, Insect, juvenile hormone analogs, Ann. licv. Riodwm. 40, 1079 (1971). 31. Suchy, I(. Slama, and F. Sorm, Inseci hormone activity of p-(1,3-Dimethylhexyl) = benzoic arid derivatives in Dystlcrc~s species, Scic~ncc 162, 382 (1968). A. W. A. Brown and I<. Pal, “Insecticide Resistance in Arthropods,” p. 2>, World Health Organization, Geneva, 1971. J. C;. Benskin and S. U. Vinson, Factors affec~ting juvenile horm~lne attalog ac.tivit y in Ndiothis
136
13.
14.
15.
16.
17.
18.
PLAPP
AND
virescens F., J. Econ. Entomol. In Press, (1972). C. E. Dyte, Resistance to synthetic juvenile hormone in a strain of the flour beetle, Tribolium castaneum, Nature (London) 238, 48 (1972). J. G. Benskin, F. W. Plapp, and S. B. Vinson, Jllvenile hormone resistance in the house fly, Paper presented at, annual meet.& Ent,omological Society of America, Los Angeles, I)er. 1971. D. C. Cerf and G. P. Georghiou, Evidence of cross-resistance to a juvenile hormone analogue in some insecticide resistant houseflies, Nature (London) 239, 401 (1972). F. W. Plapp and J. E. Casida, Genetic control of house fly NADPH-dependent oxidases: Relation to insecticide chemical metabolism and resistance, J. Econ. Entomol. 62, 1174 (1969). R. F. Hoyer and F. W. Plapp, A gross of two I)l)T-resistant genetic analysis house fly strains, J. Econ. Entornol. 59, 495 (1966). F. W. Plapp, Changes in glucose metabolism associated with resistance to DDT and
VINSON
19.
20.
21.
23.
23.
24.
dieldrin in the house fly, J. Econ. Entomol. 63, 1768 (1970). W. S. Bigley and F. W. Plapp, Choline&erase and ali-esterase activity in organophosphorussusceptible and -resistant house flies, dnn. Entomol. Sot. Amer. 53, 360 (1960). R. F. Hoyer, F. W. Plapp, and R. D. Orchard, Linkage relationships of several insertic.ide resistance factors in t,he house fly (Mlrscn domestim L.), Entomol. Exp. Appl. 8, 6.i (1963). R. D. Schonbrod, Xl. A. Q. Khan, L. C. Terriere, and F. W. Plapp, Jr., nlicrosomal oxidases in the house fly: A survey of fourteen strains, Lllfc Sci. 7, 681 (1968). M. Slade and C. H. Zibitt, Metabolism of cecropia juvenile hormone in insects and mammals, in “Insect Juvenile Hormones” (J. J. Menn and M. Berosa, Eds.), p. 155, Academic Press, New York, 1973. W. S. Bowers, Juvenile hormone: Activit.y of nat,ural and synthetic synergists, Scicnrr, 161, 895 (1968). S. J. Yu and L. C. Terriere, Hormonal modifications of microsomal oxidase activity in the house fly, Lift Sri. 10, 1173 (1971).
BIOCHEMISTRY
Juvenile
ANLI
Hormone
F.
3, 131-136
PHYSIOLOGY
ff.
liewived
Analogs: in the
PLAPP,
Novetnber
JR.,
Toxicity Housefly
AND
‘27, 1!)7:!
s.
and
BRADLEIGH
; accaepted
Cross-Resistance
l VINSON
March
LO, 197~1
The toxicity of several juvenile hormone analogs (JHAs) to susceptible and inaectitide-resistant housefly (MUSCU tlomcsticu L.) strains was det,ermined by an assay procsedure in which larvae were exposed to residues of JHAs in glass vials. All JHAs tested were toxic and the most active compound, isopropyl 11-methoxy-if, 7, ll-t,rimet,hylododeca-2, 4-dienoate, was 100 times as toxic to the susceptible Orlando Ilegular strain as methyl parat,hion and 600 times as toxic as DDT. A .i- to SO-fold tolerance to the different JHAs was present in an insecticide resistant &rain in which resistance is associated with a high level of NADPH-dependent microsomal oxidase activity controlled by a gene(s) on chromosome II. Cross-resistance was less marked in a strain wit,h a chromosome V high oxidase gene and absent in strains with ot,her resistance mechanisms. The data indicate that cross-resistance to JHAs in insects may occur in certain strains with high levels of &dative detoxifying activity. Even so, the most act.ive JHA was far more toxic t.u both susceptible a,nd resistant strains than methyl parathion or l)l)T. INTRODUCTION
Insect) juvenile hormones (JH) and their analogs (JHAs) have rccciwd a great, deal of attcwt,ion in the last scvcral years as possihlc inswt control agents. Uy disrupt.ing mc+amorphic dwrlopmcbnt, both JH and its analogs show varying dcgrws of insecticidal propcrtks. Thwcl propcrtiw arc so diffcrclnt from those of presently used synthetic organic insrct’icidcs that Williams (1) has wfcrwd to JH as a “third-genwation” Among t,hc advantagw over inwcticidr~. synthct,ic insccticidw arc wlectivity, high and thr fact that it would bc activity, difficult for an ins& to bwomc wsistant to its own hornlow. The effects of JH on diptcra wcrc first wported for AecZes aegypti 1,. (2, 3). 1 Approved 10’%7, Texas
copyright ~11 rights
for publication as Technical Art isle Agricldt.ln+al Experiment St,ation.
0 1973 by .&cademic Press. Inc. of reproduction in any form resxved.
Srivastava and Gilbrirt (4) showed t’hat JH affected pupal formation of Xarcopha!qa bdlata Parker and that th(t affrckd insects failed t,o cmc~rgc. Similar rcwlt,s \v(‘L’(’ wportcd for Roir~ft.~~stalcitrarrs (I,.) (5, 61 and for Nusca dwmestica (I,.) (7,. Sinw Williams’ statc~meiit, (1) t#hat t,hfb dwclopnwnt of rwist~ancc b.v an insect to its own hormotw n-ould bc difficult, th(t widespread assumption has been that inswts would not bccomc resistant to JH or JHA compounds. Howcvcr, since diffcrcwt. insect fatnilics vary in their response to particular JHs or JHAs (X-lo), it is possihlc t’hat diffcrenws may also occur wit,hin a spcciw. Variations in such paramctcw as penetration, metabolism, excr&~n, and/or tissue sensitivity occur bekecn inscct,icidt:susceptiblr and rwistant stra.ins (11) and thwt arc’ no good wasons why such diffw-
132
PLAPP
AND
ences should not affect hormone sensitivity as well. Several reports of tolerance to JHAs have appeared. A &fold tolerance to a JHA is present in a multiresistant strain of the tobacco budworm, Heliothis virescens F. (12), and a similar level of tolerance to a JHA occurs in a multiresistant strain of the red flour beetle, Tribotium castaneum (Herbst) (13). A tolerance to JHAs in insecticide-resistant houscflirs has bcrn described in a preliminary report from our laboratory (14) and in work by Cerf and Georghiou (15). In the latter case, tolerances of up to 40-fold were found at the LD,, level. To determine the nature of the relationship between insecticide resistance and resistance to JHAs, a number of housefly strains, with known mechanisms of resistance to insecticides, were exposed to several JHAs. By comparing the responses of these strains we hoped to determine which resistance mechanism(s) was responsible for JHA tolerance in houseflies. MATERIALS
AND
METHODS
The larvae of test housefly strains were reared on standard CSMA media. Adults were fed a 1: 1 mixture of sugar and powdered skim milk ad lib. A description of the strains used in thflse cbxpcriments follows : Orlando Regular-Insccticido-suscept,ible wild-type strain. stw; bwb; ocra-Susceptible strain homozygous for the visible recessive mutants stubby w&g, brozull body, and ocra eye, cont,rolled by genes on chromosomes II, III, and V, respectively (16). R-Fc-Resistant to DDT, carbamates, and organophosphates. Major resistance mechanism is a chromosomr V gene for high microsomal oxidase (16). R-Baygon; bwb; ocra-Resistant to carbamates, organophosphates, DDT, and cyclodienes. Major resistance genes arc chromosome II factors for high oxidase
VINSON
activity and DDT dehydrochlorinase and a chromosome IV factor for resistance to dieldrin and related compounds. The mutants b,wun body and ocra eye were introduced into the strain to eliminate resistanctx factors associated with chromosomes III and V, respectively (16). Other insecticide-resistant strains tested included Orlando DDT which has chromosome II and III genes for resistance to DDT (17), DDT-R; dov with a chromosome II gene for DDT resistance (17), kdr-0; ocra;stw with a chromosome III gene conferring resistance to DDT and pyrcthrins (17), Dld-R;cyw with a chromosome IV gene for dieldrin rcsist,ance (18), and R-Para;clw with a chromosome V altrrcd ali-&erase gene for parat#hion rrsistanrc (19, 20). Levels of oxidative detoxifying activit.y for the various resistant strains have been established previously. As measured by aldrin epoxidation in vitro, R-Baygon ;bwb ; ocra and R-F, have 2.4 and 2.0 times the activity of the st.w ;bwb ;oc.ra strain (16). In comparison with the Orlando Regular strain, aldrin epoxidasc in R-F, is four times higher than normal (21). Oxidasc activity in Orlando DDT, DDT-R;dov, kdr-0; ocra;stw, Dld-R;cyw and R-Para: clw is not significantly different from that of Orlando Regular flies (21). The JH analogs used in the tests arch described by their USDA-assigned ENT numbers and by formula. They are as follows : ENT 70033, trans-4- (6,7epoxy-3,7dimct’hyl-%octanyl)oxy-1,2-(methylenedioxybenzcne) ; ENT 70221,6,7-epoxygcranyl (p-ethyl) phenyl ether; ENT 70459, ct’hyl 3,7,1 I-trimethyldodcca-2,4-dienoate ; and ENT 70460, isopropyl 1 I-methoxy-3,7,1 ltrimethyldodeca-2,4-dienoats. Insecticides tested were DDT (technical grade) and methyl parathion (90%). EKT Nos. 70033, 70459, and 70460 were obtained from the Zoecon Corp., Palo Alto, CA. ENT No 10221 was obtained from the Stauffcr Chemical Co., Mountain View, CA. The
JUVENILE
HORMONtX
TABLE Toxicity
Data
AND
1
on the Response of Four Housejly Strains Hormone Analogs and Insecticides LCSos
Housefly
Orlando
strain
Regular
Valuea
I&l R value
stw ; bwb ; ocra
LCSO
R-Fc
LC60
R value
R-Baygon -.,~
; bwb ; ocra
R value LCjO R value
Methyl parathion 1.9a 1 3.5b 1.8 3.3b 1.7 92.6c 49
133
HOUSEFLIES
(rg/vial)
DI>T
12.6a 1 lX.Oa 1.5 154.9b 12 > 1,oooc >80
to Some Juvenile
and resistance
values
RNT 70033
ENT 70221
ENT 70459
ENT 70460
17.5a 1 81.3b 4.6 90.4b 5.1 422.5~ 24
35.3a 1 58.4a 1.6 122.4b 3.4 186.2~ 5.3
5.la I 10.9b 2.1 lO.Ob 2 34.7c 6.8
0.02a 1 O.llb - 0.r) o.lT,b 7.5 0.63~ 31
-
a LC50 values based on emergence of flies as adults. Resistance several strains to that of Orlando Regular flies. LCSo values followed different at the 95% confidence level.
DDT sample was purchased from Nutritional Biochemical Corp., and methyl parathion was obtained from American Cyanamid Corp., Princeton, XJ. The assays were performed by exposing housefly larvae to films of the test chemicals in glass vials. Mature, third (last)-instar fly larvae were collected from the rearing media 4 days after eggs were laid. Larvae of this age have usually stopped feeding as evidenced by clearing of the gut. Pupation normally occurred 24-45 hr after the tests wvcre initiated. Test solutions of all chemicals were prepared at 170 (w/v) or lower concentrations in acetone. Required amounts in 0.1 ml or less solvent were pipctt’ed with microsyringes in to glass vials of the type used for liquid scintillation counting. With additional acetone, the final volume was made up to 0.3 ml. The vials were then placed on t,hcir sides and rotated manually until the solvent was evaporated. Control vials were treated with acetone only. Twent’y larvae were placed in each vial and the top was covered with chiffon cloth and fastened with a rubber band. The vials were placed in an incubator at 27°C with a 14: 10 light: dark cycle. After 10 days t’he vials were removed and the number
values are the rat.ios of response of the by the same letter are not significantly
of flies which had successfully emerged was determined. Failure of adults to complete emergence normally was t’he criterion of assay. The results were analyzed by an IBM 360 computer program to determint IDSa values for the several chemicals in micrograms chemical/vial. In some experiments we determined the sex of emerging flies. However, since no differences relating sex to susceptibility were observed, the practice was discontinued. We also performed preliminary experiments to determine if exposure of flies to sublethal dosesof JHA resulted in a reduction in reproductive potential. Again, results were negative and the tests were discontinued. RESULTS
Data from the various bioassays are summarized in Tables 1 and 2. All results are based on a minimum of t’hree assays performed with different populations of flies and different solutions of the test materials. Table 1 presents the responses of two susceptible and two insecticide-resistant strains to all test, chemicals. The resistant strains, It-Fc and R-Baygon ;bwb ;ocra, possess resistance factors involving high levels of microsomal oxidase activity. The
134
PLAIT
TABLR Toxicity
Data
AND
2
on the Rcnponuc of Sir Strains to ENT XL@0
Housefly strain
LC:5,, (pgjvial )‘L
Orlando Regular Orlando lII>T DDT-13, ; dov kdr-0 ; ocra ; stw Did-ll; cyw It-Parathion ; clw
0.02a 0.03b O.&k 0.09c 0.01x 0.Old
Horwcfly
I(esistance value 1
2.5 4 4.5
0.5 0.5
u LCS,, values followed by the same letter are not significant)ly different at the %@,;,confidencelevel.
discussion that follows is based on the response of each strain t,o t)hc test compounds. All test materials were highly toxic to Orlando Regular house flies. Met,hyl parathion and DDT, both highly toxic insecticides, produced 50% mortalit’y at doses of approximately 2 and 12 pg/vial, respectively. Two of t.hc JHAs, ENT 70032 and ENT 70321, were less toxic than DDT by factors of 1.5- and 3-fold, rc>spcctively. ENT 70459 was intermediate in toxicity between DDT and methyl parathion. ENT 70460 was t,hc most toxic JHA tchsted. This matrrial was 100 t’imes as toxic as methyl parat,hion and 600 times as toxic as DDT. The response of the stw ;b\vb ;ocra strain followed a similar but not identical pattern t,o that of Orlando Regular flies. The major diffrrcncc \vas t,hat with all compounds tested, st,w ;bwb ;ocra larvao required 1.55.5 times as high a dose as those of Orlando Regular larvae for 50% mortalit,y. Wit.h all materials except DDT and ENT 70321 the difference in response between Orlando Regular and stw ;bwb ;ocra strains was statistically significant. This was surprising since previous tests with conventional insecticides (16) indicated that stw;bwb; ocra adults are nearly identical in response to Orlando Regular adults. Strain R-Fc, with high oxidasc activity rrsistan(*(b
to insc&c:idc~s
controlkd
by
a
gene(s) on chromosome 1’ responded simi-
VINSON
larly to t.hc stw;bwb;ocra strain to all compounds except) DDT and ENT 70221. With DDT, a lo-fold rcsistanct~ level \vas present. With ENT 70221, the LCso for R-Fc was t,wicc as great as that for stw ; bwb;ocra flies and nearly four times that of Orlando Regular flies. The R-Baygon :bwb ;ocra strain consistently showed the highcast lcvt~l of resistance t,o all chemicals tested. With methyl parathion and DDT, rcbsistancc levels were 4%fold and greater than 80-fold, respectively. Wit,h the JHAs, the dose Ievrls required for 50% mort,alitg n-erc 5- to 30-fold greater than for Orlando Regular flies. Resistance was highrst with EN’1 70321 and ENT 70460. In both cast’s th(l LCsO valurs wclre 20- to 80-times greater t,han for Orlando Regular flies and fivchtimes great,er than for stw;bwb ;ocra flks. In all cases, LCsas were significantly higher for this st’rain than for all othcbrs. Additional tests were’ condu&d with ENT 70460 on several othcbr insc&cido resistant strains. The r(ksults (Table 2) indicate this JHA is highly act#ive against all
test
fly
strains.
Tht:
compound
\vas
more toxic to dieldrin- and parathionresist,ant strains than to Orlando Regular flks. The three DDT-resistsant strains w(bre only slightly less susceptible. It is worth not,ing that none of the strains list>ed in Table 2 possess high levc~ls of micrtrsomal oxidasc act,ivity as a component of thrir rcsistanre spectra. DISCUSSION
The data show that some JHAs are highly toxic to houseflies treated as larvae and that the most active compound, ENT 70460, is far more toxic than standard insccticidcs. The dat,a also confirm previous findings (12-15) of c~ross-rc~sist’ancc, to JHAs in insecticide-resistant st#rains. Our result’s show that cross-resistance is highest in the R-Baygon ;bwb ;ocra strain which has chromosomfl II g;c~nc~s for high microsomal oxidascb acativity and DDT dehydrochlorinase as well as a chromosome
.TI:VENIT,E:
HORMONES
IV gene for cyclodiene resistance. Since other strains with the DDT dehydrochlorinase- and cyclodicnc-resistance genes were susceptible to the test chemicals, it is likely t#hat# the chromosome II high oxidaso gcnr is responsible for rcsistancc~. Ilouseflies possessing this factor havcl previously been shown to mctabolizca man) irisc&c*idcn, i.cb., aldrin, allrthrin, caarbaniatrs, and organophosphat,c~s at grcbatcr rates than suscrptibh: flies (16). The reason for the relatively higher rclsistance to JHAs of R-Baygon;bwb ;ocra as compared with R-Fc is not’ known. Both strains have high oxidase activit,y as measured with aldrin epoxidasc>. HOWWW. nit,h other insccticidc chemical substratcls, differences exist brtwccn tht> st#rains. For rhxamplc, It-Baygon ;bwb ;ocra Aics metabolize allethrin brttcr than R-Fc flies while with DDT thr convcrsc is true (10). Thus, qualitative diff (rcnces exist, bctwecln th(k strains and th(ly art’ apparcnt)ly rcflcrtc~d in diffcrcnces in rc’sponscb to JH.4s as well. Published data (22) on th(l role of hydroxylation and cipoxidatjion in hormonc~ mc%abolism support the hypot’hesis that microsomal oxidases arc important, in JHA mc+abolism. Th(rc is at present, no adequat,(x biochemical rvidcncc that this microsomal oxidasck systc>ni is involvf)d in rrsistancc. lIowev(r, thcb cxar1ic.r report, (23) of JH activit,y of microsomal oxidascl inhibit,& synrrgists and tht> recent finding (24) that JlIAs arc microsomal oxidasc inducers further implicate the system as of prime importance in JHA toxicity. The occurrence of cross-resistance to JHAs in the housefly strains resistant’ to insc&cidcs suggests t)hat c’xposure to prcscnt~ly used toxicants may lrssc~l the potential of JHAs as control agmlts. F’urthrrmort>, this c~xperimr~ntally induccbd cross-rcsist,ance to JHAs implies that even highc~r 1~~~1s of rcsistancc may develop wl1c.n ins& populations arc exposed to horuion(~s in cont,rol programs. If this is tru(a, th(>n the use of JH and JHA materials may not. be the solution to resistance
AND
135
HOVSEFLIES
problems originally hoped for. However, it is worth noting that even with resistance bating prctsclnt, the most’ active JHA is morcx toxic to housefly larvae of bot)h susceptible and resistant strains than either DDT or mc+hyl paratjhion. ACKNOWLEDGlIENTS
We thank Ms. E. A. Butler for technical assistance during the course of these experiments. 1)r. J. (+. Benskin, formerly of this laboratory, participated in a number of preliminary experiments which led to this publication. REFERENCES
1. C.
2.
:<.
4.
5.
6.
7.
6.
9. 10.
11.
12.
hl. Williams, Third-generation pesticides. Insect hormones and their analogs could provide highly selective insecticides, Sri. Amrr. 117, 13 (1969). A. Spielman and C. M. Williams, Lethal effects of synthetic juvenile hormone on larvae of the yellow fever mosquito, A&s aegypti, Scimcr 154, 1043 (1966). A. Spielman and V. Skaff, Inhibition of metamorphosis and of ecdysis in mosquitoes, J. Znwrt Phgsiol. 13, 1087 (1967). V. S. Srivastava and L. I. (Gilbert, Juvenile hormone : Effects on a higher dipteran, Scicncc~ 161, 61 (196X). J. E. Wright, Hormones for control of livestock arthropods. Development of an assay to select candidate compounds with juvenile hormone activity in the stable fly, J. Econ. Entornol. 63, x7x (1970). J. Ji:. Wright. and G. h:. Spates, Biological evaluation of juvenile hormone rompounds against pupae of the stable fly, J. Agr. Food Chew. 19, 289 (1971). A. Herzog and B. E. Monroe, Effect of synt.hetic juvenile hormone and citric acid on housefly pupae, ,II usca domrstica I,., Camp. RiochwL. Phz/siol. 41, 481 (19723. J. J. Glenn and IVI. 13eroza, “InsecBt Juvenile Hormones : Chemistry and Action,” 341 pp., Academic Press, New York, 1972. 6. Slama, Insect, juvenile hormone analogs, Ann. licv. Riodwm. 40, 1079 (1971). 31. Suchy, I(. Slama, and F. Sorm, Inseci hormone activity of p-(1,3-Dimethylhexyl) = benzoic arid derivatives in Dystlcrc~s species, Scic~ncc 162, 382 (1968). A. W. A. Brown and I<. Pal, “Insecticide Resistance in Arthropods,” p. 2>, World Health Organization, Geneva, 1971. J. C;. Benskin and S. U. Vinson, Factors affec~ting juvenile horm~lne attalog ac.tivit y in Ndiothis
136
13.
14.
15.
16.
17.
18.
PLAPP
AND
virescens F., J. Econ. Entomol. In Press, (1972). C. E. Dyte, Resistance to synthetic juvenile hormone in a strain of the flour beetle, Tribolium castaneum, Nature (London) 238, 48 (1972). J. G. Benskin, F. W. Plapp, and S. B. Vinson, Jllvenile hormone resistance in the house fly, Paper presented at, annual meet.& Ent,omological Society of America, Los Angeles, I)er. 1971. D. C. Cerf and G. P. Georghiou, Evidence of cross-resistance to a juvenile hormone analogue in some insecticide resistant houseflies, Nature (London) 239, 401 (1972). F. W. Plapp and J. E. Casida, Genetic control of house fly NADPH-dependent oxidases: Relation to insecticide chemical metabolism and resistance, J. Econ. Entomol. 62, 1174 (1969). R. F. Hoyer and F. W. Plapp, A gross of two I)l)T-resistant genetic analysis house fly strains, J. Econ. Entornol. 59, 495 (1966). F. W. Plapp, Changes in glucose metabolism associated with resistance to DDT and
VINSON
19.
20.
21.
23.
23.
24.
dieldrin in the house fly, J. Econ. Entomol. 63, 1768 (1970). W. S. Bigley and F. W. Plapp, Choline&erase and ali-esterase activity in organophosphorussusceptible and -resistant house flies, dnn. Entomol. Sot. Amer. 53, 360 (1960). R. F. Hoyer, F. W. Plapp, and R. D. Orchard, Linkage relationships of several insertic.ide resistance factors in t,he house fly (Mlrscn domestim L.), Entomol. Exp. Appl. 8, 6.i (1963). R. D. Schonbrod, Xl. A. Q. Khan, L. C. Terriere, and F. W. Plapp, Jr., nlicrosomal oxidases in the house fly: A survey of fourteen strains, Lllfc Sci. 7, 681 (1968). M. Slade and C. H. Zibitt, Metabolism of cecropia juvenile hormone in insects and mammals, in “Insect Juvenile Hormones” (J. J. Menn and M. Berosa, Eds.), p. 155, Academic Press, New York, 1973. W. S. Bowers, Juvenile hormone: Activit.y of nat,ural and synthetic synergists, Scicnrr, 161, 895 (1968). S. J. Yu and L. C. Terriere, Hormonal modifications of microsomal oxidase activity in the house fly, Lift Sri. 10, 1173 (1971).