Effects of Sublethal Dosages of Insecticides on Culex

JUNE 1989 Sust,nrtrlr, Erprcrs oF INsEcrrcrDEs

EFFECTSOF SUBLETHAL DOSAGESOF INSECTICIDESON CULEX QUINQUEFASCIATUSI

L. L. ROBERT2'3ANo J. K. OLSON,

ABSTRACT. Groups of Culex quinquefasciatuswere exposed as fourth instar larvae to sublethal concentrations (0.1 LCsoand-LCsJ of malathion, methoprene,propoxur or resmethrin. Femalesexposed as larvae, to an LCuolevel of methoprene had reduced wing length and longevity. Egg production was reduced by 5p% and 39Voin tho-semosquitoes exposed to t-C- levels of malathidn" or methoprene, resp-ectively.In contrast, egg production and egg raft size increasedfollowing treatment with 0.i LCu; levels of malathion or methoprene.^Femalesexposed as larvae to methopren6 laid,S0% fewer eggspe"i raft, and egghatching decreased36% comparedwith controls. _- Femalesexposed as larvae to LC56levels of malathion or methoprenelaid shorter eggsthan controls. The proportion of femalesin the adult population was reducedfollowing exposureto e'iiher propoxur or resmethrin, and increasedfollowing exposureto malathion. Time to pupation and time of emergenceof the adult populations were increasedfollowing exposureto most of the insecticidal treatmentj. These results indicated that a single, sublethal exposure to certain insecticides had a simificant effect on mosquito reproduction.

INTRODUCTION These reports indicate that in certain cases, sublethal effects of pesticidesmay be as impor- The acute toxicity of insecticides is used to tant as the acute toxic effect in reducing num- produce mortality in target insect populations bers of insect pests. Knowledge of these suble- inside a given area.Treatment of a target species thal effects ofpesticides on arthropods is there- with an insecticidemay be uneven due to factors fore important to the decision making processes such as vegetation or wind, with some individ- in integrated pest managementprograms. uals therefore receiving sublethal dosagesof in- The purpose of this study was to investigate secticides.Sublethal dosagesof insecticides may the effectsof selectedinsecticides LCso and 0.1 affect insect populations by: 1) affecting sur- LCsolevels) upon the reproductive potential of vival, 2) affecting the reproductive ability of Culcx quinquefasciatusSay, an important urban individuals or 3) affecting the genetic makeup vector of diseaseagents in the southern United of future generations(Moriarty 1969). States.The following parameterswere measured Before World War II it was known that arsen- to determine the effects of sublethal concentra- ical and botanical insecticideshad sublethal ef- tions of insecticides on the insecticide-exposed fects on insects (Hoskins 1940). At the time, and Fr generations;adult longevity, adult wing these sublethal effects were viewed as inconse- length (a measureof adult body size),number quential. Subsequentwork with DDT, botani- of eggs per raft, egg length, number of eggs cals and cyclodienes has been reviewed by produced per female, egg hatching, sex ratio of Ascher(1964) and Moriarty (1969).Other insec- adults and developmentaltime. ticides have been shown to affect a number of reproductive parameters in mosquitoes,includ- MATERIALS AND METHODS ing fecundity (Firstenberg and Sutherland 1981, Kelada et al. 1981,Kerdpibule et al. 1981,Liu The UTMB strain of Cx. quinquefasciatus, et al. 1986),egg hatching (DeCourseyand Webs- colonized at the University of Texas Medical ter 1952, DeCourseyet al. 1953), immature de- Branch, Galveston, was used in this study. An velopment time (Wijeyaratnea),adult longevity insecticide-free colony of this strain has been (Duncan 1963) and adult size (Wijeyaratnea, maintained at Texas A&M University for over Keladaet al. 1981). 10 years. It was determined to be susceptibleto the insecticidesused in this study. 1 This researchwas conducted in cooperation with Mosquitoeswere rearedat 27'C,75-85VoRH the Agricultural Research Service, USDA and ap- with a 14:10hr light/dark photoperiod. T$o egg proved for publication as TA 2422I by the Director of rafts (ca. 200 eggsper raft) were placed in white the Texas Agricultural Experiment Station. enamelpans (36 x 23 X 5 2 cm) containing 1.0 Department of Entomology, Texas A&M Univer- liter of deionized water. Larvae hatching from sity, CollegeStation, TX77843. 3 these eggswere fed a diet consisting oflab chow, Present Address: Department of Entomology, lactalbumin and brewer's yeast (1:1:1 ratio). Walter ReedArmy Institute of Research,Washington, quantitatively (Gerberg DC 20307-5100. Food was added et al. a Wijeyaratne, P. M. 19?6. Effects of sub-lethal 1969) starting on the day after the eggs were Iarvicide exposureon population and flight character- placed into the pans. Pupae were transferred to istics of Culexpipiens quinquefascintus.Ph.D. Disser- a beaker of water and placed in a wood and tation. Univ. of Texas Health ScienceCenter at Hous- screencage (50 cm). The adults emergedand fed ton. 245 pp. od libiturn on l07o sucrosesolution. The blood 240 JouRNlr- oF THE AunRrcen Moseumo CoNrnol AssocrerroN Vol. 5, No. 2 meal source consisted of a restrained chick Table 1. Mean LCso(ppb), LCso(ppb) and slope placed in the cage3 days after emergence. values for Cubx quin4u,efascintuslawae exposed to The following insecticideswere used: 1) mal- insecticides,serially diluted with acetonein 100 ml athion (95.9%) (American Cyanamid Co., of deionizedwater.* Wayne, NJ); 2) propoxur (99.0%) (Mobay Chemical LCuo LCso Slope Kansas MO); 3) resmeth- ChemicalCorp., City, 35.0 51.0 3.6 (86.8%) (Penick-Bio malathion rin UCLAF Corp., Lyn- methoprene 0.1 0.5 2.3 dhurst, NJ); and 4) methoprene(89.25%) (Zoe- propoxur 287.0 439.0 6.6 con Corp., Dallas, TX). Acetonewas used as a resmethrin 4.3 7.0 5.9 solvent to dilute all chemicalsto the appropriate * Averageof 3 consistent replicates. concentrations. Immatures were exposedto the various insec- 2. Mean (+96; per female and early fourth instar larvae. Controls for Table number of eggs ticides as eggsper laft fot Culcx quinqucfasciatusexposed as each experimental group were treated with ace- larvae to insecticides. tone and handled in the same manner as the insecticide exposed groups. Fifby larvae were Number of eggsper female placedin 100ml of deionizedwater in a 355-ml Control 0.1 LCEo LCso disposableplastic cup. Those larvaetreated with G.C.1 Insecticide group group group malathion, resmethrin or propoxur were exposed malathion 132(15) 141(11) 66 (19)* for a 24-hr period without food. After exposure, methoprene 183 (15) r94 (25) 111 (19)i the larvae were removed from the insecticide propoxur 153 (e) L44(r4) 168 (16) solution, rinsed, placed in fresh water and fed resmethrin 146 (12) 136(2) 148 (4) Iarval diet. Larvae exposedto methoprene were kept in the insecticidal solution until adult 2 malathion r28 (7) 88 (7)* 94 (5)* emergencewith food added after the first 24-hr 2 methoprene 203(11) 161(25)r 143(5)** period. This procedure was followed because 2 propoxur 198 (29) 161(23) r73 (7) mature fourth instar larvae are most susceptible 2 resmethrin r24 (6) 130(5) 117 (10) to juvenile hormone mimics (Spielman and Eggsper raft Skaff 1967) and exposure during this period is critical. Control 0.1 LC6o LCao group group group Three days after emergence,replicates from G.C.r Insecticide eachinsecticide treatment consistingof 3 groups malathion 144 (8) 156 (8) r52 (7) of 10 males and 10 femaleswere placed in wood methoprene 215 (10) 227 (r2) 150 (8)* (5) (5) (4) and screen cages (30 x 22 x 22 cm). Females propoxur 204 202 1e3 resmethrin 152 (8) 160 (7) 174 (6) were given the opportunity to feed on a restrained chick. Egg rafts were collected daily. 2 malathion 165 (8) 157(8) 155 (7) The procedurewas repeatedafter 8 daysto allow 2 methoprene 200 (7) 205 (5) 155 (8)+ forthe completion ofa secondgonotrophic cycle. 2 propoxur 198 (8) 194(6) r79 (7) Analysis of variance was performed using the resmethrin 151(8) 156(7) 171(5) ANOVA and GLM procedures, depending on * Value is significantly different from control, P < whether the experiment involved a balanced or 0.01. unbalanced design (SAS 1985). The data from ** Value is sigrificantly different from control, P < the egg hatching studies, expressedas percent- 0.001. I ages,were transformed using the arcsin angular Indicates first or secondgonotrophic cycle. transformation to normalize the distribution before testingbv ANOVA (Sokaland Rohlf 1981). mortality indicated resmethrin was the most Individual meanswere comparedusing least sig- toxic (LCso : 4.3 ppb) to larvae, followed by nificant difference (LSD). This procedure was malathion (LC50 : 35 ppb) and propoxur (LCso used because the comparison-wise error rate : 287 ppb). These LCso values are generally from each pairwise comparison was considered consistent with those previously reported to be more important than the experiment-wise (Wijeyaratnen, El-Khatib and Georghiou 1985). error rate (Jones1984). This allowsall possible The highest concentration of methoprene comparisonsbetween all experimental groups. used was not lethal to larvae but was highly effective in preventing adult emergence, as evi- RESULTS AND DISCUSSION denced by the low LCso (0.1 ppb). The LCso value for methoprene was similar to that re- The insecticides used in this study varied in ported earlier for Cx. quinquefasciatus (Schaefer their effectivenessin killing early fourth instar and Wilder 1972). larvae of Cx. quinqu.efasciatus(Table 1). Com- Egg production was significantly reduced (P parison of the 3 insecticideswhich cause Iarval < 0.01) during the first gonotrophic cycle by JUNE 1989 Sust,urHnr,Errrcrs or INsocrrcrops 241

50Vo and 40Vo, and,in the second gonotrophic ious concentrations of insecticideswhich inhib- cycle by 30% and,4O%following exposureto the ited the emergence of 25% of adults had an LC56 treatment of malathion or methoprene, increasein basalfollicle numbers (Saleh and Aly respectively(Table 2). In contrast,the 0.1 LCso 1987,Saleh 1988).Sutherland et al. (1967)in- treatments of malathion or methoprene each dicated that maximum sublethal dosages of had a slightly stimulatory effect on eggproduc- DDT, dieldrin and malathion resulted in signif- tion in the first gonotrophic cycle. Both concen- icantly increasedbasal follicle number in female trations of malathion and methoprene resulted Ae. aegypti. In contrast, Firstenberg and Suth- in significantly reduced (P < 0.01) egg produc- erland (1981) reported decreasedfecundity in tion during the secondgonotrophic cycle. How- Ae. aegyptipopulationsfollowing exposureto 0.1 ever, none of these effects were observedin the LC"6 dosagesof malathion or temephos. Fr generation. Conflicting results as to the effects of insec- Other investigators have reported reducedegg ticides on mosquito eggproduction appear to be production following larval exposure to suble- related to the dosagesused. Our data (and that thal concentrations of insecticides. Fecundity of most of the studies cited) indicate that fe- was reduced in Cx. quinquefasciatw following males which are exposedas larvae to selecting larval exposure to selecting concentrations of doses(causing some mortality in the population) dimilin, methoprene, dichlorvos, malathion of insecticideshave reducedegg production and (Wijeyaratnea)or temephos (Ferrari and Geor- femalesexposed as larvae to nonselectingdoses ghiou 1981). Various insecticides also reduced (causinglittle or no mortality) may have either eggproductionin Aedesaegyprl (Linn.) (Firsten- increasedor decreasedegg production (Table 3). bergand Sutherland198L) and Cx.pipiensLinn. Although some discrepancies occur, selecting (Gaabouband Dawood1974). dosesof insecticidesgenerally increaseegg pro- The slight increase in egg production during duction in adult mosquitoes. the first gonotrophic cycle following exposureto The LC"o treatment of methoprene resulted 0.1LCuo treatments of either malathionor meth- in eggrafts that were significantly smaller (P < opreneis also consistent with somestudies using 0.01)than thoseproduced by controls(Table 2). low (causing little or no mortality) insecticidal However, the 0.1 LC56 treatment resulted in dosages.Culcxpipiens females that survivedvar- slightly more eggsper raft than controls during

Table 3. Summary of previous researchon eggproduction in adult mosquitoesfollowing larval exposureto selectingand nonselectinginsecticidal doses.

Chemical Dose Species Effect* Reference Selectingdoses Altosid LCuo-* Ae. aeglpti _EP Naqvi et al. t976 methoprene LCuo Ae. aegypti -EP Firstenberg and Sutherland 1981 Abate Penfluron 0.1-1.0ppb An. steplwnsi -EP Saxenaand Kaushik 1986 PuryItriazine 5-10 ppm DDT 20 ppm Cx. quinEtefasciatus -EP Kerdpibule et al. 1981 temephos LCs, LCso Cx. quirquefasciahts -EP Ferrari and Georghiou 1981 malathion LC- Cx. quinqtnfasciatw -EP Wejeyaratnea dimilin dichlorvos methoprene DDT LC. Cx. pipiens _EP Gaabouband Dawood 1974 Altosid 0.1-1.0ppb Cx. pipi.ens +FN Gaaboubet al. 1981 dimilin IGRs ICrt Cx. pipiens +FN Saleh and Aly 1987 chlorpyrifos IC"u Cx. pipi.ens +FN Saleh1988 sumithion

Norcelectirry d.oses DDT 0.1ppm Ae. a.eglpti +EP Havertz and Curtin 1967 6IGRs sublethal Cx. pipiens +EP Kelada et al. 1981 methoprene 0.1 LC5o Ae. ocgypti -EP Firstenberg and Sutherland 1981 Abate temephos 0.01 ppm Cx. quirquefasci,atus _EP Ferrari and Georghiou 1981 DDT, dieldrin, ma]- sublethal Ae. a,egypti +FN Sutherland et al. 1967 athion

JUNE 1989 Sust-ntHer, Eprncrs on Ilsnctlcrops 243

Table (tSE) 5. Mean length of eggsfrom Cubx quin4u.efasciatusfemales exposed as larvae to insecticides. Mean egglength (rnm) Control 0.1 LCs G.C.1 Insecticide group group LC56 group malathion 0.71(0.03) 0.70(0.04) 0.66(0.03)** methoprene 0.72(0.02) 0.71(0.03) 0.67(0.03)** propoxur 0.71(0.02) 0.71(0.02) 0.70(0.02) resmetrin 0.72(0.03) 0.70(0.03) 0.70(0.03) 2 malathion 0.73(0.03) 0.71(0.03) 0.69(0.02)** 2 methoprene 0.69(0.01) 0.68(0.02) 0.67(0.01)" 2 propoxur 0.70(0.02) 0.70(0.01) 0.70(0.02) 2 resmethrin 0.69(0.02) 0.6e(0.02) 0.69(0.02) * Value is significantly different from control, .P < ** 0.01. Value is significantly different from 'Indicates control, .P < 0.001. first or secondgonotrophic cycle.

Table (+gB1 6. Mean adult wing length and longevity of Cubx quin4uefasciaarcexposedas larvae to insecticides,

Wing length (mm) Control 0.1 LCm Sex Insecticide group group LC6e group M malathion 2.29(0.03) 2.31(0.03) 2.37(0.05) M methoprene 2.61(0.02) 2.71(0.03)r 2.59(0.02) M propoxur 2.65(0.01) 2.66(0.01) 2.63(0.03) M resmethrin 2.42(0.06) 2.36(0.04) 2.36(0.02)

F malathion 2.87(0.03) 2.85(0.03) 2.82(0.05) F methoprene 3.19(0.03) 3.22(0.03) 3.10(0.03)* F propoxur 3.04(0.01) 3.04(0.03) 3.05(0.01) F resmethrin 2.84(0.06) 2,77(0.08) 2.84(0.021 Adult longevityr Control 0.1 LCso Sex Insecticide group group LC66 group M malathion 23.0(3.0) 22.3(0.7) 2r.0(r.2\ M methoprene 26.3(2.3) 20.3(1.8)* 16.3(2.8)* M propoxur 2r.0(2.5) 26.3(4.5) 2r.7(2.2) M resmethrin 18.3(1.3) 19.3(0.9) 20.0(2.r) F malathion 28.7(2.7) 2e.6(3.2) 29.0(1.0) F methoprene 28.0(1.0) 25.0(3.1) 16.0(4.4)* F propoxur 28.7(4.8) 30.7(1.5) 31.7(2.0) F resmethrin 22.0(L.0) 25.7(3.5) 24.3(3.0) *Value is significantly different from p I control, < 0.01. Expressedas LToo,the number of days to bd% mortality. reported detrimental insecticidal effects on in- of methoprene had sigtrificantly shorter wings sect ovarioles and ovariole development (Zagh- (P < 0.01). In contrast, Wijeyaratnea reported Ioul and Brown 1968, Judson and de Lumen that larval treatments with methoprene had no 1976, Saleh 1988), none reported any effect on effect on wing length. However, Wijeyaratnea egg length. did find that dimilin, dichlorvos and malathion Adult wing length was influenced as a result treatments cause reduced wing length in fe- of exposure to methoprene (Table 6). Males males. Conversely, Kelada et al. (1981) reported exposed to the 0.1 LCso treatment of methoprene that LCso treatments of 6 IGRs, including meth- had significantly longer wings (P < 0.01) than oprene, caused increased wing length in female controls. Females exposed to the LCuo treatment Cx. pipiens. They also found that the LCro treat- 244 JouRNALoF THE AIranmceNMosqurto Conrnol Assoclenol VoL.5,No.2

Table ?. Mean (+951 days to pupation and adult emetgence of Culex quinquefasciatus exposed as latvae to insecticides.

Emergence time2

Chemical Dose Pupation time' Males Females Control 0 0.7 (0.06) 2.3(0.20) 2.7 (0.27)

malathion 0.1 LCso 1.1(0.12)* 3.0(0.25)* 3.1(0.15)* malathion LCuo 0.9(0.11)* 2.4(0.07) 2.9 (0.r4)

methoprene 0.1 LCso 1.0(0.14)* 2.6(0.16)* 3.2(0.18)* methoprene LCro 0.9(0.11)* 2.9(0.23)* 3.0(0.14)*

propoxur 0.1 LCso 0.9 (0.08)* 2.4(0.r7) 2.5(0.10) propoxur LCuo 0.7 (0.09) 2.5(0.10) 2.7 (0.20)

resmethrin 0.1 LCso 0.9 (0.07)* 2.7(0.t4)* 2.7 (0.06) resmethrin LCro 0.9 (0.08)* 2.6(0.14)* 2.6(0.10) t Time (days) from insecticidal treatment to pupation. 'Time (days) from insecticidal treatment to adult emergence. * Value is significantly different from control, P < 0.05. ment of methoprene resulted in reduced female emergewas greater than controls for the mala- wing length. Abu-Hashishu,using LC'o and LCzs thion, methoprene and resmethrin treatments. levels of 6 synthetic pyrethroids, reported no Femalesof the populationsexposed to the 0.1 significant changes in wing dimensions of Cr. LCs6treatments of malathion or to either treat- pipiens. ment of methoprene also took a significantly Only methoprene-exposedmosquito popula- longer time (P < 0.05) to emergethan did con- tions showed a significant reduction (P trols (Table 7). Increased Iarval and pupal pe- < 0.01) in longevity compared with controls riods resulting from insecticidal treatments is (Table 6). Both exposurelevels of methoprene consistent with the earlier research of resulted in reduced longevity in males and fe- Wijeyaratnea who reported that LCso levels of males.This result contrasts with the findings of dimilin, dichlorvos, malathion and methoprene Wijeyaratnea who suggests that methoprene prolong the larval development period of Cr. doesnot affect longevity of Cx. quinquefascintus. quin4uefosciatlus.Lengthened lawal develop- However, Arias and Mulla (1975),working with ment time was reflected in an increasedgener- Cx. tarsalis Coq. and the chemicals methoprene ation time, particularly with dimilin and meth- and dimilin, found decreasedadult mortality oprene. This pattern was also reported with rates after larval exposureto 0.1 and 1.0 ppb. various dosagesof other insecticides in autoge- Larval exposureto malathion, propoxur and nous Cr. pipiens (Farghalu). resmethrin did not sigrrificantly affect adult lon- Adult sex ratios were affected by exposureto gevity (Table 6). Likewise, larval exposure to methoprene,propoxur or resmethrin (Table 8). malathion did not affect adult longevity in C.r. The proportion of adult females in the insecti- pipiens (Gaabouband Dawood 1974), Cx. quin- cide-exposedpopulations were signifi cantly re- quefasciatus(Wijeyaratnea) or Ae. oegypti (Fh- duced (P < 0.01) following either propoxur or stenberg and Sutherland 1981). Sublethal dos- resmethrin exposure. In contrast, methoprene agesof DDT (Gaabouband Dawood 1974,Kerd- exposureresulted in a significantly increased(P pibule et al. 1981) and resmethrin (Focks 1984) < 0.01) proportion of femalesin the insecticide- did not affect adult longevity in various mos- exposedand untreated Fr population. These ef- quito species. fects were not observed in the Fz population. Except the LCro treatment of propoxur, both Wijeyaratneaalso observedsimilar sex ratio ef- concentrations of all insecticidesresulted in sig- fects associatedwith methoprenetreatments on nificantly Iengthened (P < 0.05) time to pupa- Cx. quin4uefasciotus.Gradual increasesin meth- tion (Table 7). The time needed for males of oprene concentrations were likewise accom- insecticide-exposed mosquito populations to panied by gradual increasesin female:maleratio in autogenousCx. pipiens (Farghal and Temerak 1981). 5Abu-Hashish, T. A. 1978.Morphological and bio- logical studies in Egyptian mosquitoesunder the ac- tion of synthetic pyrethroids with a new order of 6Farghal, A. I. 1979. Recent studies in culicine activity. Ph.D. Thesis. Faculty of Agriculture, Univer- mosquito control. Ph.D. Thesis. Assuit University, sity of Alexandria, Egypt. Esvpt.