Studies on the Mechanism of DDT Resistance in Culex pipiens fatigans

Bull. Org. mond. Sante' 1970, 42, 623-629 Bull. Wid Hitha Org.J Studies on the Mechanism of DDT Resistance in Culex pipiens fatigans Lipids of Susceptible and Resistant Strains R. L. KALRA1 The mechanism of DDT resistance in Culex pipiens fatigans is poorly understood. Earlier studies indicated that the dehydrochlorination ofDDT does not explain resistance in this species. Studies on the role of lipids as a mechanism of resistance included the estimation of lipid content and the determination of the proportions of different classes of lipids in the larvae of susceptible and resistant strains. There was no evidence of any correlation between the lipid content andDDT resistance in this species and the proportions of neutral lipids, phospholipids and fatty acids of different strains did not indicate any consistent correlation with DDT resistance. Within one strain, the larvae containing the higher amount of lipids were able to resist better the toxic effect ofDDT. Analysis offatty acids of the larvae that survived and died as a result of treatment with DDT did not reveal any difference. Neither p,p'-DDT nor o,p'-DDT at sublethal concentration affected the lipids of the larvae of susceptible and resistant strains. Culex pipiens fatigans, the vector of Brancroftian filariasis, has been found to be DDT-resistant almost all over the world.2 However, the mechanism of DDT resistance in this species is poorly understood (Busvine, 1967). Kalra et al. (1967) and Hooper (1968) observed that the metabolism of DDT to DDE does not explain resistance in this species. Increased lipid content as a mechanism of DDT tolerance has been studied in a number of insects. Munson (1953), Munson & Gottlieb (1953) and Munson et al. (1954) investigated the possible relationship between lipid content and DDT toler- ance in cockroaches and considered that lipids act as a competitive site for DDT that prevents the insecticide from reaching its usual site of action. Wiesmann (1957) reported that a DDT-resistant strain of housefly contained more lipids than normal strains. However, a comparison of several resistant 1 Research Officer, National Malaria Eradication Pro- gramme, 22 Alipore Road, Delhi, India. ' Pal, R. & Kalra, R. L. (1965) Survey of insecticide resist- ance in Culicine mosquitos; unpublished document, WHO/ Vector Control/122.65. 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 and susceptible strains of houseflies revealed no correlation between lipid content and resistance (Ascher & Neri, 1961). Neri et al.3 observed that DDT-selected Anopheles atroparvus and Anopheles stephensi had significantly greater fat contents than the parent strains from which they had been selected. In Aedes aegypti, the DDT-resistant strain was not characterized by an increased lipid content (Fast & Brown, 1962). Numerous attempts have also been made to correlate the nature of fatty acids, iodine value and sterol content with the tolerance of insects to insecticides (Fast & Brown, 1962; Moore et al., 1967; Ascher & Neri, 1961; Enan et al., 1964). The present investigation was undertaken to elucidate the role of lipids, if any, in DDT resistance in C. p. fatigans. The nature of neutral and phospholipids present in this species has, however, already been reported by Kalra et al. (1969). Neri, I., Ascher, K. R. S. & Mosna, E. (1959) Etudes sur les Anophelines resistant aux insecticides. 5. Contenu en lipides de la femelle d'Anopheles atroparvus; unpublished document, WHO/Mal/249. A limited number of copies of this document is available to persons officially or pro- fessionally interested on request to Distribution and Sales, World Health Organization, 1211 Geneva, Switzerland. 2504 -623- R. L. KALRA MATERIALS AND METHODS Insect material Susceptible and DDT-resistant strains of C. p. fatigans were used. The susceptible strain (Cali- fornia-S) was obtained by the National Com- municable Diseases Center, Savannah, Ga., USA, from the University of California, Riverside, Calif., USA, and was then transferred to the National Institute of Communicable Diseases (NICD), Delhi. Delhi-N strain is being maintained without any intentional contamination with the insecticide. The DDT-resistant strain originated in Rangoon (Ran- goon-R) and was first selected at the Department of Zoology, University of Western Ontario, London, Canada, and a subcolony was obtained from there. This strain was maintained at the NICD, Delhi, under continuous selection with p,p'-DDT. Larvae of a strain designated as Delhi-R were originally collected from sprayed areas around Delhi and were maintained in the laboratory under selection pressure with o,p'-DDT. All the strains were maintained following the methods described by Krishnan (1964). Wherever results with different strains are compared the larvae were reared under parallel conditions. Extraction of lipids The larvae were blotted on filter-papers and were then weighed and homogenized with 20 volumes of chloroform-methanol (2: 1, v/v) for 2 min in a Waring Blendor at room temperature. The homo- genate was allowed to stand for 2 hours and was then filtered. After filtration, the chloroform-methanol extract was purified following the method of Folch et al. (1957). The total lipid content was determined gravimetrically and the phosphorus in the lipids was estimated by the method of Bartlett (1961). Fractionation and estimation of neutral and phospholipids Lipids were fractionated into neutral and phospho- lipids by silicic acid column chromatography (Kalra et al., 1969). Cholesterol in the neutral lipid fraction was estimated by the Liebermann-Burchard method (Huang et al., 1961). Neutral lipids were further fractionated by thin-layer chromatography on plates coated with silica gel G using petroleum ether/ ether/acetic acid (90: 10: 1) as the solvent and the amounts of glycerol corresponding to the tri- glycerides, diglycerides and monoglycerides were estimated by the method of Van Handel & Zilversmit (1957). Phospholipids were fractionated by thin-layer chromatography on plates coated with silica gel G using chloroform-methanol-water (65 : 25 : 4; v/v/v) and chloroform-methanol-7 N ammonia (65: 25: 4; v/v/v) solvents and the individual phospholipids were identified and estimated (Rao et al., 1967). Characterization offatty acids The total lipids fraction was transmethylated using 6% methanol-sulfuric acid (Feldman & Rouser, 1965). The methyl esters of the fatty acids were extracted with distilled hexane and characterized by gas-liquid chromotography, using the Perkin-Elmer gas chromatograph model F-Il (single-column, equipped with flame-ionization detector). The stationary phase consisted of 1: 4 butanediol succinate supported on Chromosorb W. The identifi- cation of the methyl esters of the fatty acids was done by comparing their relative retention time with those of methyl esters of standard fatty acids (Farquhar et al., 1959) and the area under each peak was measured to calculate the relative proportion of the different fatty acids. RESULTS The lipid contents of the susceptible and resistant strains of C. p. fatigans is given in Table 1. The lipid content of the larvae of California-S strain was found to be 30 mg/g of wet tissue, and phospholipids constituted about 40% of this amount. Cholesterol was, however, found only in traces. DDT-resistant strains from India did not differ from the California-S strain in either the amount of total lipids or the amount of phospholipids. The larvae of Rangoon-R strain were found to contain significantly greater amounts of total lipids than other susceptible and resistant strains. The difference was, however, not evident in the amounts of phospholipids and cholesterol. Thin-layer chromatography revealed that tri- glycerides constituted most of the neutral lipids of the larvae in all the strains of C. p. fatigans studied. The estimation of glycerol indicated that about 96%-98 % of the glycerol occurred as triglycerides. Diglycerides, monoglycerides and free fatty acids were present only in traces. There was no difference in the pattern of neutral lipids between the different strains. The phospholipids, as characterized by their mobility and staining behaviour on plates coated with silica gel G, were found to be polyglycerol 624 MECHANISM OF DDT RESISTANCE IN C. P. FATIGANS: STUDY OF LIPIDS TABLE I LIPIDS OF SUSCEPTIBLE AND RESISTANT STRAINS OF C. P. FATIGANS a I CoLC Total Total TotalLCSilD o L lipids phospholipids cholesterolStrain of pp'-DDT of dieldrin (m/g (mg/g (mg/gj(ppm) (ppm) wet tissue) wet tissue) wet tissue) California-S 0.03 0.13 30.3 ± 0.6 12.2 i 0.8 0.61 i 0.09 Delhi-N 0.6 0.14 30.9 ± 1.5 11.7 + 0.8 0.65 i 0.09 Rangoon-R >40.0 0.24 37.8 ± 1.6 11.7 i 0.5 0.88 i 0.11 Delhi-R >40.0 >0.50 29.6 ± 1.5 12.3 i 0.9 0.61 ± 0.07 "P " values Comparison between: California-S and Delhi-N >0.50 >0.50 >0.50 California-S and Rangoon-R <0.02 >0.20 >0.10 California-S and Delhi-R >0.50 >0.50 >0.50 a The results are based on 3 replicates and expressed as the mean + the standard error. TABLE 2 PERCENTAGE COMPOSITION OF PHOSPHOLIPIDS IN SUSCEPTIBLE AND RESISTANT STRAINS OF C. P. FATIGANS a Phospolipid ] California-S 1 Delhi-N 1 Rangoon-R Delhi-R Polyglycerol phosphatide 5.1 ± 0.6 8.3 ± 1.5 4.2 ± 0.3 7.7 ± 1.5 Phosphatidyl ethanolamine 53.8 ± 3.3 50.2 ± 4.2 54.1 ± 2.5 52.1 i 3.5 Phosphatidyl choline 23.1 ± 1.2 24.7 ± 2.5 22.0 ± 1.0 21.3 ± 2.2 Phosphatidyl inositol 7.2 ± 2.3 8.5 ± 2.4 6.6 ± 1.1 7.4 ± 1.3 Phosphatidyl serine 8.1 ± 2.7 5.7 ± 1.3 10.2 ± 3.8 7.3 i 1.3 Unidentified lipid 2.4 + 0.2 2.3 + 0.3 2.8 ± 0.6 4.2 ± 1.2 a The results are based on 3 replicates and expressed as the mean ± the standard error. phosphatide, phosphatidyl ethanolamine, phos- phatidyl choline, phosphatidyl serine and phosphat- idyl inositol. Another ninhydrin-positive phospho- lipid, as yet unidentified, was also detected. No plasmalogens of acid-alkali-stable phospholipids were found. The results indicated that both the nature and amount of the phospholipids present were almost the same in the susceptible and resistant strains of C. p. fatigans (Table 2). The major fatty acids found in the larvae of C. p. fatigans were myristoleic acid, palmitic acid, palmitoleic acid, stearic acid and oleic acid. Traces of lauric acid, myristic acid and linoleic acid were also detected in the larvae. The proportions of the major fatty acids in the larvae of different strains is given in Table 3. The California-S, Delhi-N and Delhi-R strains ofC.p.fatigans contained the different fatty acids almost in the same proportions, whereas in the Rangoon-R strain there were slightly higher amounts of oleic acid. It is, however, quite evident that the proportions of fatty acids in the different strains did not reveal any consistent pattern with regard to their tolerance to DDT. Late third-instar larvae of the Delhi-N strain were exposed to 1.0 ppm ofp,p'-DDT, the concentration that caused about 60% mortality after 20 hours 625 R. L. KALRA TABLE 3 FATTY ACID COMPOSITION OF THE LARVAE OF SUSCEPTIBLE AND RESISTANT STRAINS OF C. P. FATIGANS a Fatty acids | Call- T Delhi-N Rangoon- Delhi-Rfornia-S - R Myristoleic acid 6.1 5.7 0.8 6.2 Palmitic acid 28.7 25.4 27.1 29.1 Palmitoleic acid 23.7 26.5 38.0 27.8 Stearic acid 25.8 24.9 24.8 25.1 Oleic acid 15.6 16.8 9.3 11.3 a Only the major fatty acids were estimated and the values for each are expressed as a percentage of the total. The results are based on the average of 2 replicates. exposure. The survivors and dead larvae were pooled separately and their total lipid and phospho- lipid contents were estimated. For comparison, estimates were also made of the lipids of untreated larvae kept under identical condition in distilled water without insecticide. The results revealed that the surviving larvae contained significantly greater amounts of total lipids than the dead and untreated larvae (Table 4). The amount of phospholipids was also significantly greater in the survivors than in the dead and untreated larvae. After a short exposure to p,p'-DDT estimation of the lipids of knocked-down and unaffected larvae TABLE 4 LIPIDS OF SURVIVING OR KILLED LARVAE OF THE DELHI-N STRAIN AFTER TREATMENT WITH p,p'-DDT AND OF UNTREATED LARVAE a Totallipids TotalT(mgg wet tissue)p ( phospholipids(m g u mg/g wet tissue) Survivors 22.6 i 0.5 5.2 i 0.3 Dead 6.7 ± 0.5 2.7 i 0.2 Untreated 16.2 0.1 4.6 0.1 P " values Comparison between: Survivors and dead <0.001 <0.01 Survivors and untreated <0.02 <0.10 a The results are based on 3 replicates and are expressed as the mean ± the standard error. indicated that the unaffected larvae contained greater amounts of lipids, as shown below: Total Phospho- lipids lipids Unaffected 48.0 12.4 Knocked-down 26.2 6.3 The proportions of fatty acids of the surviving and dead larvae that had been exposed to DDT were the same and did not differ significantly from those of the untreated larvae given in Table 3. To study the possibilities of lipogenesis as a result of treatment with DDT, the lipids of the larvae of susceptible and resistant strains were estimated after exposure to sublethal concentration of p,p'-DDT and o,p'-DDT (Table 5). The results indicated that p,p'-DDT even at the concentration of 10.0 ppm did not increase or decrease the lipids in the larvae of the highly resistant strains Rangoon-R and Delhi- R. There was no indication that the larvae of the Delhi-N strain accumulated more lipids after exposure to 0.5 ppm of p,p'-DDT for 4 hours. However, the amount of lipids in the larvae of Delhi-N strain had decreased after exposure to p,p'-DDT for 20 hours, as shown below: Total lipids Phospholipids Lipids content (mg/g wet weight) Treated Untreated larvae larvae 18.4+1.3 24.2+0.5 6.9+0.5 8.4±0.2 P values <0.02 0.05 The lipids of treated and untreated larvae of the California-S strain did not reveal any significant difference after exposure to p,p'-DDT. Exposure to o,p'-DDT did not alter the lipids either. DISCUSSION The results obtained did not indicate any correla- tion between the lipid content and DDT resistance in C. p. fatigans. The larvae of the Rangoon-R strain were observed to contain significantly higher amounts of lipids than the susceptible strain, but another resistant strain, Delhi-R, contained as much lipids as the susceptible strain. Fourth-instar larvae of C. p. fatigans attained a total lipid content of about 3% of the wet weight. Fast & Brown (1962) found that the lipids constituted about 3.7% of the wet weight of the larvae of Aedes aegypti. Phospho- lipids constituted about 40% of total lipids of C. p. fatigans, and the same proportion was found in the larvae of Ae. aegypti (Fast & Brown, 1962). Periplaneta americana, however, contained about 33% as phospholipids (Siakotos, 1960). 626 MECHANISM OF DDT RESISTANCE IN C. P. FATIGANS: STUDY OF LIPIDS TABLE 5 EFFECT OF DDT ON THE LIPIDS OF SUSCEPTIBLE AND RESISTANT STRAINS OF C. P. FATIGANS a Treated larvae Untreated larvae Strain itsectiidlse Total Phospho-i Total Phospho-Strain intreatmenta lipids lipids I lipids lipids(mg/g wet (mg/g wet (mg/g wet (mg/g wet tissue) tissue) tissue) tissue) California-S a p,p'-DDT 21.7 5.0 23.3 4.5 0.5 ppm for 2 h (±1.1) (±0.4) (±0.9) (±0.2) Delhi-N a p,p'-DDT 34.2 8.7 38.6 9.7 0.5 ppm for 6 h (±1.9) (±0.6) (±2.7) (±0.8) Rangoon-R a p,p'-DDT 38.1 8.0 35.9 7.0 2.5 ppm for 20 h (±3.0) (±0.6) (±1.4) (±0.4) Delhi-R b p,p'-DDT 25.9 6.3 24.8 5.2 10.0 ppm for 20 h Delhi-N b o,p'-DDT 31.3 9.6 31.2 9.7 0.5 ppm for 4 h Delhi-R a o,p'-DDT 35.5 7.8 36.7 7.1 10.0 ppm for 20 h (±2.4) (±0.9) (±3.5) (±0.5) a Based on 3 replicates and expressed as the mean ± the standard error (in parentheses). b Based on average of 2 replicates. Mullins (1954) postulated that DDT exerted its toxic effect against the insects by interference with the cell membrane. It was therefore considered possible that alterations in the cell membrane may render the insect less sensitive to the action of DDT. As phospholipids are an important constituent of cell membranes, and have also been implicated in the active transport of sodium ions (Hokin & Hokin, 1959), any alteration in the cell membrane structure may be reflected in the phospholipid content: no such difference was found, however, between susceptible and resistant strains of C. p. fatigans. This observation is in agreement with that of Fast & Brown (1962) who also did not observe any difference in the phospholipid pattern of susceptible and resistant strains of Aedes aegypti. Bridges & Ricketts (1967) were successful in changing the pattern of phospholipids in the housefly, by rearing them on synthetic diets, but could not find any difference in their sensitivity to the action of insecticides. Never- theless, Bridges et al. (1962) found differences in the turnover of 32p in different phospholipid frac- tions in dieldrin resistant and susceptible strains of houseflies and considered these to be an indication of an alteration in the membrane permeability. Preliminary experiments carried out with larvae of the Delhi-N and Delhi-R strains of C. p. fatigans, which differed considerably in their resistance to DDT, have not revealed any significant difference either in the metabolism of 32P or the pattern of labelling of the phospholipids in these two strains (Kalra, unpublished). Fatty acids with from 12 to 18 carbon atoms were found in C. p. fatigans whereas Buffington & Zar (1968) observed fatty acids with from 15 to 20 carbon atoms in C. p. pipiens. The relative proportion of saturated and unsaturated fatty acids was found to be the same in susceptible and resistant strains. Munson (1953), however, showed that the cock- roaches with a smaller proportion of saturated fats were less susceptible to DDT. Ascher & Neri (1961) reported no consistent difference in the iodine value of the lipids of susceptible and resistant strains of houseflies. Fast & Brown (1962) also did not find any difference in fatty acids in relation to DDT resistance in Aedes aegypti. Our results indicated, however, that among a single batch of larvae the individuals that survived the exposure to DDT did show a higher total lipid content than those that died. The smaller amount of lipids in the dead larvae was not merely due to degeneration of lipids as the difference was apparent between the knocked-down and unaffected larvae after a short exposure to DDT. The results therefore suggest that the larvae containing a higher amount of lipids were able to resist better the toxic effect of 627 R. L. KALRA DDT. Reiser et al. (1953), and more recently Moore et al. (1967), showed that weevils surviving the toxic effect of insecticides usually contained more lipids. Bennett & Thomas (1963) also observed that the susceptibility of the alfalfa weevil to insecti- cides decreased as the amount of body fat de- creased. The pattern of fatty acids in the surviving and dead larvae did not reveal any difference bet- ween them in contrast to the findings of Moore et al. (1967) who observed higher levels of stearic, linoleic and linolenic acids in the dead weevils and higher levels of palmitic and oleic acids in the survivors. Hoskins & Gordon (1956) and Gordon (1961) considered that a higher amount of lipids may give a small amount of protection to insects but that this may not have any bearing on the high specific resistance generally manifested by them. These factors, if co-existent with other defence mechanisms, however, may ultimately result in the manifestation of markedly high tolerance to insecticides (Spiller, 1958; Winteringham & Hewlett, 1964). McLean & McLean (1966) observed that DDT and phenobarbital showed similar behaviour in inducing the synthesis of certain enzymes in rat liver. The effect of DDT on enzyme induction has now been well established even in insects (Agosin et al., 1965). Remmer & Merker (1963, 1965) showed that phenobarbital administration to rats induced a massive increase in the smooth membrane and its components, e.g., protein, lipid and RNA, in the liver. It was, therefore, considered possible that DDT might affect the intermediary metabolism of resistant strains in such a way that they could synthesize higher amounts of lipids thus enabling them to survive the toxic effect of DDT. However, the untreated larvae of the resistant strains of C. p. fatigans were found to contain as much lipid as those exposed to DDT. No increase in the amount of lipids was observed even when the larvae were reared from the second stage onwards in a p,p'-DDT suspension. From the foregoing discussion it seems that the lipids do not play any role in the resistance of C. p. fatigans to DDT. The resistant strains used in the present investigation were also dieldrin- resistant. It therefore appears that the lipids are not implicated even in dieldrin resistance in this species, in contrast to the findings of Khan & Brown (1966), who observed the higher amount of lipids in the dieldrin-resistant strains of Aedes aegypti. ACKNOWLEDGEMENTS It is a pleasure to acknowledge the guidance offered by Professor T. A. Venkitasubramanian, Head of the Department of Biochemistry, V. B. Patel Chest Institute, University of Delhi, in the course of this work. Thanks are due to Dr N. G. S. Raghvan, Director, National Institute of Communicable Diseases, India, and Dr S. L. Dhir, Director, National Malaria Eradication Programme, India, for their interest in the work. Thanks are also due to Dr Albert S. Perry, Scientist- Director, National Communicable Diseases Center, Savannah, Ga., USA, and Professor A. W. A. Brown, former Head of the Department of Zoology, University of Western Ontario, London, Canada, for making available the California-S and Rangoon-R strains of C. p. fatigans for this investigation. The author gratefully acknowledges the technical assistance rendered by Shri Sunder Singh and is indebted to Mr Francis of the Shri Ram Institute of Industrial Research, Delhi, India, for his help in carrying out the gas-liquid chromatography. RURESUME ETUDE DU MECANISME DE LA RESISTANCE AU DDT CHEZ CULEXPIPIENS FATIGANS: LIPIDES DE SOUCHES SENSIBLES ET RESISTANTES Le r6le des lipides dans le m6canisme de la r6sistance au DDT a et etudie chez quatre souches de Culex pipiens fatigans presentant differents degres de sensibilite a l'insecticide. Apres extraction au chlorofoi me-methanol, les lipides de larves au 4e stade ont ete fractionnes par chromatographie en lipides neutres, phospholipides et acides gras. Le contenu en lipides des larves equivalait a environ 3% de leur poids humide. On n'a decouvert aucun indice d'une correlation entre cette teneur et la r6sistance au DDT. Chez toutes les souches, les lipides neutres etaient essentiellement representes par des glyc6rides. On a identifie divers phospholipides: phosphatidyl-ethanola- mine, phosphatidyl-choline, phosphatidyl-inositol, phos- 628 MECHANISM OF DDT RESISTANCE IN C. P. FA TIGANS: STUDY OF LIPIDS 629 phatidyl-serine, phosphatide polyglycerolique, et decele la presence d'un lipide non encore determine. Toutes les souches contenaient des proportions quasi identiques de ces differents composes. Les acides gras portaient de 12 a 18 atomes de carbone et aucune correlation n'a pu etre etablie entre leur repartition et la resistance au DDT. Parmi les larves d'une meme souche, les specimens ayant la plus forte teneur en lipides ont le mieux resiste a l'action toxique du DDT. L'analyse des acides gras chez les larves survivantes et les larves tuees, apres une exposition au DDT, n'a pas montre de differences entre souches sensibles et souches resistantes. Des concentra- tions subletales de p,p'-DDT et de o,p'-DDT n'ont provoque aucune modification de la teneur en lipides des larves de souches sensibles ou resistantes. 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