Results of fenitrothion selection of Culex pipiens fatigans Wied. and Anopheles albimanus Wied*

Bull. Org. mond. Sante 1969, 40, 97-101Bull. Wid Hith Org. Results of Fenitrothion Selection of Culex pipiens fatigans Wied. and Anopheles albimanus Wied.* G. P. GEORGHIOU 1 & J. R. CALMAN 2 Laboratory selection ofCulex pipiens fatigans (=C. quinquefasciatus) and Anopheles albimanus by fenitrothion pressure for 30 and 25 generations, respectively, resulted in only 2.2-fold and 1.1-fold increases in tolerance to this compound. Cross-resistance to arprocarb in Culex increased 12.2-fold but smaller increases in tolerance to fenthion, malathion, DDT, and dieldrin, may be manifestations of vigour tolerance. Since the essential conditions for selection are believed to have been fulfilled, the authors consider the negative results obtained to be ofpractical significance, as well as ofacademic interest. Among several organophosphates which have recently attracted attention for use in mosquito control, fenitrothion [= Sumithion, OMS-43, o-o-dimethyl o-(4-nitro-m-tolyl) phosphorothionate] is characterized by a pronounced degree of selectivity: its mammalian toxicity is reported to be low (LC50 oral for rats, 250 mg-670 mg per kg of body-weight (Kenaga, 1966)) while its toxicity to mosquitos is relatively high (LC50 for Culex fatigans larvae, 0.02 ppm). The field performance of this compound is being investigated in connexion with the WHO Malaria Eradication Programme (Bar-Zeev & Bracha, 1965; Vandekar, 1965; Coz et al., 1966). Past experience dictates that the introduction of a new compound in large-scale insect control opera- tions should be preceded by an investigation of the resistance potential of the target species towards the compound. This fact has prompted us to study the possibility of development of resistance to fenitrothion in 2 species of mosquitos, Culex pipiens fatigans Wied. (= C. quinquejasciatus Say) and Anopheles albimanus Wied. Although our carefully conducted selections did not lead to resistance to fenitrothion and, therefore, have not yielded information on the character of resistance to this compound in mosquitos, we consider the results to be of practical significance, as well as of academic * This study was supported in part by Public Health Service Research Grant CC 00301 from the National Com- municable Disease Center, Atlanta, Ga., USA. 1 Associate Professor of Entomology, University of California, Riverside, Calif., USA. 2 Laboratory Technician, University of California, Riverside, Calif., USA. interest, and they are reported briefly in the present paper. MATERIALS AND METHODS Two stock strains were utilized in these selections: (1) the " Coachella" strain of C. fatigans collected in 1963 in the Coachella Valley of southern Cali- fornia; at the time of its colonization, the strain was moderately resistant to DDT and also exhibited low levels of tolerance to a number of organo- phosphates and to dieldrin (Georghiou, Metcalf & Gidden, 1966); (2) the " Haiti " strain of A. albi- manus, collected in 1965, resistant to dieldrin and moderately resistant to DDT.3 The 2 selected strains are referred to as " Culex-fenitrothion" and "Anopheles-fenitrothion ". The 2 parental strains together with our sus- ceptible laboratory strain of C. fatigans have pro- vided base-line levels of susceptibility with which the results of selection can be compared. Our rearing method for C. fatigans, the selection and testing techniques, and the treatment of data, were described in a previous paper (Georghiou, Metcalf & Gidden, 1966); the A. albimanus colonies were handled in the same manner, except that the adults were fed on rabbits. The selection of C. fati- gans was based entirely on larval pressure. In 3 Georghiou, G. P. & Gidden, F. E. (1965) Anopheles albimanus from Haiti; susceptibility to insecticides and selectionfor resistance to OMS-33 and OMS-43 (unpublished working document WHO/VC/187.65). A limited number of copies of this document is available to persons officially or professionally interested on request to Distribution and Sales, World Health Organization, 1211 Geneva, Switzerland. 2279 97- G. P. GEORGHIOU & J. R. CALMAN TABLE I CHANGES IN SUSCEPTIBILITY TO FENITROTHION IN LARVAE OF C. FATIGANS DURING SELECTION BY FENITROTHION Strain and generation Fenitrothion190 d II_____tested LCso (ppm) 95 %fiducial limits Slope (b) ± SE (b) RR Laboratory-1964 0.022 0.020-0.025 7.84 ± 1.20 - Coachella-1964 (Pi) 0.020 0.018-0.022 6.95 ± .82 0.9 Cu/ex-fenitrothion F. 0.017 0.015-0.018 8.63 i 1.05 0.8 Fg 0.015 0.013-0.016 9.99 i 1.30 0.7 F. 0.019 0.011-0.025 8.89 ± 2.46 0.9 F4 0.022 0.014-0.027 6.70 ± 1.66 1.0 F. 0.025 0.024-0.026 8.94 i 0.67 1.1 F. 0.023 0.023-0.024 8.43 i 0.51 1.0 F. 0.017 0.016-0.017 7.98 ± 0.57 0.8 F,o 0.029 0.026-0.031 8.43 i 0.87 1.3 Fie 0.059 0.055-0.066 9.26 ± 1.22 2.7 F25 0.056 0.054-0.057 14.11 i 1.14 2.5 F30 (1966) 0.048 0.047-0.049 8.62 + 0.43 2.2 Coachella-1966 (control) 0.014 0.014-0.015 8.06 ± 0.90 a RR= resistance ratio, i.e., LCso of selected strain/LCso of laboratory strain. A. albimanus, the selection pressure was on larval stage of generations F1 to F20 and on adult stage of generations F21 to F25. the the RESULTS AND DISCUSSION Selection of C. fatigans by fenitrothion pressure was begun in September 1964 and continued until September 1966. Altogether, 30 generations were completed, of which 28 were selected and 2 were left unselected in order to avoid a severe reduction of the population. Dosage-mortality regression lines (ld-p lines) were determined on the parental and on 11 filial generations, as shown in Table 1. Although the susceptibility of the strain appeared to fluctuate somewhat from generation to generation during selection, it did not decrease appreciably, having reached in F3. a level only 2.2-fold lower than it was initially. The susceptibility of the parental, control strain also did not change significantly during the same period (Table 1). As in the larvae, susceptibility of adults changed little in the selected strain, i.e., the LC50 of F30 adults was 6.7 'ug/cm2 compared with an LCr0 of 3.2 Lg/cm2 in the susceptible strain. The cross-resistance spectrum of the selected strain underwent changes that may be mostly manifestations of vigour tolerance (Table 2). Thus, there were 4-fold and 4.4-fold increases in LC50 values for fenthion and malathion, respectively, without appreciable change in the slope of the Id-p lines. Also, the LC50 value for dieldrin increased 9.1-fold, as a result of considerable increase in the slope of the ld-p line which remained within the original range of dosages characteristic of the parental population. The most important increase in cross-resistance was towards the carbamate arprocarb (= Baygon), which amounted to 12.2-fold at the LC,0 level. This increase could not be attri- buted to contamination from our arprocarb- selected strain since in other respects, especially in their response to DDT, the 2 strains were distinctly different. It should also be noted that there was an increase in the LC,0 value for DDT from an 98 FENITROTHION SELECTION IN CUL.EX PIPIENS FATIGANS AND ANOPHELES ALBIMANUS TABLE 2 PATTERN OF SUSCEPTIBILITY TO INSECTICIDES IN LARVAE OF FENITROTHION-SELECTED STRAIN AND 2 NONSELECTED STRAINS OF C. FAT GANS Laboratory strain: Coachella strain (Pi): Culex fenitrothion strain: Compound tested LCso in ppm LCso in ppm RRa LCso in ppm RRa(and slope b ± SE) (and slope b ± SE) (and slope b ± SE) Fenitrothion 0.022 (7.84 ± 1.20) 0.02 (6.95 ± 0.82) 0.9 0.048 (8.62 ± 0.43) 2.2 Fenthion 0.0077 0.0091 (7.62 ± 0.57) 1.2 0.031 (7.41 ± 0.93) 4.0 Malathion 0.081 (10.82 ± 1.02) 0.124 (5.85 ± 0.44) 1.5 0.36 (7.46 ± 0.56) 4.4 Arprocarb 0.23 (5.27 ± 0.25) 0.26 (3.29 ± 0.20) 1.1 2.8 (6.48 ± 0.38) 12.2 DDT 0.026 (4.87 ± 0.28) 0.11 (2.18 ± 0.11) 4.2 0.78 (2.06 ± 0.34) 30.0 Dieldrin 0.0076 (3.94 ± 0.28) 0.0083 (3.37 + 0.39) 1.1 0.069 (9.35 ± 0.58) 9.1 a RR = resistance ratio, i.e., LCso of strain studiedlLCso of laboratory strain. initial level of 0.11 ppm to 0.78 ppm without any change in the slope of the Id-p line (Table 2). Although this may have been the result of selection, its significance is in doubt since DDT tolerance also rose during the same period in the unselected Coachella strain, the LC50 value at the end of this study being 1.59 ppm (b=0.99±0.08). The selection of A. albimanus by fenitrothion pressure extended from September 1965 to March 1967 and involved 25 generations. The effects of selection on susceptibility to fenitrothion and other compounds were less pronounced than in C. fatigans, and are shown in Table 3. The non-development of resistance during labo- ratory selection must not be regarded as an indica- tion that the species as a whole lacks resistance potential towards the compound under study. It is believed, however, that if the following conditions are fulfilled, negative results may be considered significant: (1) the parental strain is of recent origin and derived from a large number of individuals collected from different localities to ensure a rich genetic background; (2) the rearing method pre- serves the genetic variability of the strain; and (3) the insecticidal selection pressure is not so severe as to cause the elimination of characters that are essential in the stabilization of fully expressive resistant genotypes. These conditions have been carefully observed in the present selections. It is of interest that similar selection by DDT and by arprocarb of substrains from the same stock popula- tion of C. fatigans did yield strains resistant to these TABLE 3 PATTERN OF SUSCEPTIBILITY TO INSECTICIDES IN LARVAE OF A FENITROTHION- SELECTED STRAIN AND OF THE PARENTAL STRAIN OF A. ALB MANUS Haiti strain (P.): Anopheles-fenitrothion strain: Compound tested LCso in ppm LC5o in ppm RRa(and slope b + SE) (and slope b ± SE) Fenitrothion 0.046 (8.02 ± 0.77) 0.052 (5.84 ± 0.71) 1.1 Fenthion 0.029 (6.72 ± 0.59) 0.029 (3.15 ± 0.70) 1.0 Malathion 0.32 (3.28 + 0.27) 0.38 (7.88 ± 0.98) 1.2 Arprocarb 0.37 (9.90 ± 0.64) 0.54 (10.90 ± 0.82) 1.5 DDT 0.029 (3.19 ± 0.30) 0.033 (1.72 i 0.10) 1.1 Dieldrin (10 ppm) 50 % mortality 30 % mortality - a RR = resistance ratio, i.e., LCs. of selected strain/LCso of Haiti strain. 99 G. P. GEORGHIOU & J. R. CALMAN TABLE 4 SUSCEPTIBILITY TO FENITROTHION IN LARVAE OF STRAINS OF C. FATIGANS AND A. ALBIMANUS SELECTED BY VARIOUS INSECTICIDES LCso in ppmLCoipm Strain tested limits) for seective RRb (and 95 % fiducial limits) RRb compound a frfntoho C. faiigans Susceptible - 0.022 (0.020-0.025) - DDT-resistant Fit 6.39 (5.28-7.67) 246.0 0.024 (0.022-0.026) 1.1 Arprocarb-resistant F35 5.78 (5.58-5.99) 25.1 0.039 (0.036-0.043) 1.8 A. albimanus Susceptible - 0.046 (0.044-0.049) - DDT-resistant F30 0.21 (0.19-0.23)C 191.0 Dieldrin-resistant F4 3.30c 1 650.0 0.038 (0.036-0.040) 0.8 Arprocarb-selected F2o 1.06 (1.01-1.10) 2.9 0.051 (0.045-0.058) 1.1 a Name of selective compound indicated in strain designation. b RR = resistance ratio, i.e., LCso of selected strain/LCso of suceptible strain. c LC5o values for S strain in Georghiou & Metcalf (1963). compounds. Thus the level of DDT-resistance increased from a 4.2-fold level to a 256-fold level in 11 generations, and the level of arprocarb resistance reached a 25-fold level in 35 generations (Geor- ghiou, Metcalf & Gidden, 1966). In A. albimanus, fully resistant substrains were obtained by dieldrin pressure within 4 generations, and high levels of resistance to DDT within 25 generations. It is noteworthy that we have also observed little or no fenitrothion cross-resistance in strains selected with other insecticides. Table 4 indicates the levels of susceptibility to fenitrothion in strains of C. fati- gans and A. albimanus following their selection by DDT, dieldrin and arprocarb pressure. The lack of resistance to fenitrothion in A. albimanus may not be surprising; no resistance to organophosphates or carbamates has yet appeared in field or laboratory populations of Anopheles. But the lack of response to fenitrothion selection in C. fatigans must be viewed with the knowledge that resistance to other organophosphates and carbamates did appear in Culex. ACKNOWLEDGEMENTS The authors are grateful to Mrs Frances E. Gidden, Mr Zafar M. Iqbal, Mrs Ethel Ban and Mrs Ruth Millholin, for technical assistance at various phases of these investigations. REISUME RESULTATS DE LA SILECTION DE CULEX PIPIENS FA TIGANS WIED. ET D'ANOPHELES ALBIMANUS WIED. PAR LE FtNITROTHION Etant donne le caractere tres s6lectif de la toxicite du fenitrothion a l'egard des mammiferes et des insectes et l'interet qui s'est recemment manifest6 au sujet de son emploi comme insecticide, les auteurs ont examine la possibilite d'apparition de la resistance 'a ce produit chez deux especes de moustiques. La selection au laboratoire de Culex pipiens fatigans (= C. quinquefasciatus) et d'Anopheles albimanus par le 100 FENITROTHION SELECTION IN CULEX PIPIENS FATIGANS AND ANOPHELES ALBIMANUS 101 tenitrothion, pendant 30 et 25 generations respectivement, n'a abouti qu'a une augmentation de la tolerance de 2,2 fois et 1,1 fois. La resistance crois6e i I'arprocarb (Baygon) chez Culex s'est accrue de 12,2 fois; on a observe, dans une mesure moindre, des accroissements de la tol6rance au fenthion, au malathion, au DDT et a la dieldrine, mais on peut les considerer comme resultant d'une tolerance de vigueur. Etant donn6 que les conditions essentielles a la selection ont et6 observees, les auteurs estiment que les resultats negatifs qui ont 6te obtenus presentent un interet pratique aussi bien que theorique. REFERENCES Bar-Zeev, M. & Bracha, P. (1965) Bull. Wld Hlth Org., 33, 461 Coz, J., Venard, P., Attiou, B. & Somda, D. (1966) Bull. Wld Hlth Org., 34, 313 Georghiou, G. P. & Metcalf, R. L. (1963) Science, 140, 301 Georghiou, G. P., Metcalf, R. L. & Gidden, F. E. (1966) Bull. Wld Hlth Org., 35, 691 Kenaga, E. (1966) Bull. ent. Soc. Amer., 12, 188 Vandekar, M. (1965) Bull. Wld Hlth Org., 33, 107