Susceptibility status of transgenic Aedes aegypti (L.) against insecticides W.A. Naznia#, S. Selvia, H.L. Leea, I. Sadiyaha, H. Azaharia, N. Derricb and S.S. Vasanc a Medical Entomology Unit, Infectious Disease Research Centre, Institute for Medical Research, Jalan Pahang 50588 Kuala Lumpur, Malaysia b
Oxitec Limited, 71 Milton Park, Abingdon, Oxford OX14 4RX,UK c
CEBAR, IPS Building, Level 5, Block B, University of Malaya, Kuala Lumpur, 50603, Malaysia Malaysia Oxitec S/B, Plaza See Hoy Chan, Suite 1502, Jalan Raja Chulan, Kuala Lumpur, 50200, Malaysia
Abstract Two strains of Aedes aegypti, a RIDL® strain (MyRIDL513A) and a laboratory strain (MyWT), were used in the insecticide-susceptibility study. Two-to-five-day-old female mosquitoes from both strains were tested for their susceptibility against seven insecticides from the class of organophosphate, carbamate, pyrethroids and organochlorine. The adult bioassay was performed according to WHO standard procedures. The 50% lethal time (LT50) value was determined for each strain against the seven insecticides. Both the MyRIDL513A and MyWT strains were resistant to DDT, exhibiting mortality of 48% and 33% respectively, but were susceptible to malathion (5%), permethrin (0.75%), cyfluthrin (0.15%) and lambdacyhalothrin (0.05%). This study shows there is no evidence of altered susceptibility to insecticides in the RIDL strain compared to a WT strain of Ae. aegypti. Keywords: Aedes aegypti; transgenic Ae. aegypti; dengue; insecticides; RIDL Ae. aegypti.
Introduction Dengue is a mosquito-borne infection that in recent decades has become a major international public health concern. Dengue is prevalent in the tropical and sub-tropical regions of the world, predominantly in urban and semi-urban areas. Dengue was
first reported in Malaysia in 1901–1902 on the island of Penang[1] and has since spread nationwide. A total of 30 981 cases and 70 deaths were recorded in Malaysia up to September 2009.[2] Ecological, behavioural and control information on population size, distribution, survivorship, seasonal abundance and insecticide susceptibility is urgently
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required for an understanding of the epidemic potential and for the formulation of new control strategies. Control and/or elimination of mosquitoes is a complex and difficult challenge but it remains the only viable option in the absence of a vaccine or antiviral treatment. Although space-spraying of insecticides is used widely, this method has so far failed to control the spread of Aedes aegypti , and long-term implications such as resistance[3] and the effects of residues in the environment are important considerations. Recent advances in molecular biology have brought some exciting new control possibilities.[4,5,6] A particular technology known as RIDL® (Release of Insects carrying Dominant Lethality) has been developed in Ae. aegypti and, while the bionomics of transgenic Ae. aegypti have been evaluated and found to be indistinguishable from the wild type,[3] it is necessary to continue evaluating other aspects of the biology of RIDL Ae. aegypti as Ae. aegypti has been found to be resistant to insecticides in numerous locations throughout the world.[7] This paper reports the susceptibility status of the RIDL Ae. aegypti against insecticides commonly used to control the vector mosquitoes.
United Kingdom, and reared in the Arthropod Containment Laboratory Facility (ACLF) in the Institute for Medical Research, Kuala Lumpur.
My RIDL-513A strain The original RIDL Ae. aegypti strain was designated as LA513A and was generated in a Rockefeller strain genetic background. The strain used in this study was generated (laboratory strain of Malaysian origin using 12 homozygous female founder parents) by out-crossing to the MyWT strain. The RIDL strain was maintained in the Arthropod Containment Level-2 (ACL-2) laboratory at 26±1 °C and 70%–80% relative humidity with a photoperiod of 10 hours of artificial daylight and 14 hours of darkness.[8] Ten drops of Liquifry® No. 1 fish food were added to induce egg hatching in a tray half filled with 1.5 litre of tetracycline water (tet-water) at 30 mg/l to suppress the lethal effect of the RIDL system. Larvae were fed with powdered fish food (Tetramin®) while emerged adults were fed with 10% sucrose supplemented with 1% vitamin B complex solution soaked in lint cloth and placed inside a small plastic bottle. Five days after adult emergence, the females were permitted to blood-feed on mice. Three days after feeding, a piece of moist filter paper in a porcelain bowl half filled with water was introduced for oviposition.
Materials and methods Mosquito strains Two strains of mosquitoes were used, viz. a laboratory-susceptible strain designated as MyWT strain originating from Penang, Malaysia, and reared in the laboratory for 1014 generations since 1965, and an F2 RIDL strain obtained from Oxitec Limited,
MyWT strain The origin of the MyWT strain was from Selangor, a state in peninsular Malaysia. The MyWT larvae were reared as above, except that the first and second instar larvae were fed
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on cow liver powder whereas the third and fourth instar larvae were fed on small pieces of partially-cooked liver. Emerged adults were fed with 10% sucrose solution supplemented with 1% vitamin B complex solution soaked in lint cloth and placed inside a small plastic bottle.
diagnostic dosages at the respective exposure period. [9] Cumulative mortality counts or knockdown were recorded at regular intervals for the respective exposure periods. After the experiment, all mosquitoes were transferred into a clean paper cup and provided with 10% sugar solution. The test mosquitoes and the controls were held for a 24-hour recovery period and mortality was recorded. If the control mortality was between 5% and 20%, the percentage mortalities were corrected by Abbott’s formula.[10] All data were analysed using a probit analysis computer programme and LT50 and LT90 for each insecticide for both the strains were calculated.[11]
Insecticides The insecticides used in the adult susceptibility test were diagnostic dosages as specified in the WHO standard method. The insecticideimpregnated papers were obtained from the Vector Control Research Centre, Universiti Sains Malaysia. The adults were tested against two organophosphates (5% malathion, 1% fenitrothion), three pyrethroids (0.75% permethrin, 0.05% lambdacyhalothrin, 0.15% cyfluthrin), an organochlorine (4% DDT) and a carbamate (0.1% propoxur). The exposure time for DDT was half an hour and the exposure time for all other insecticides was one hour.
Results and discussion The RIDL strain was originally developed in a Rockefeller background, which is a laboratory strain originally isolated from the wild over 50 years ago. We wanted to potentially improve this strain by out-crossing to a more recently isolated strain. This was done into a Malaysian strain isolated from the wild more than 45 years ago. In order to determine that the process of out-crossing had no effect on the strain background in terms of insecticide resistance, it was tested against several insecticides commonly used for control. According to WHO,[12] if the mortality is in the range of 98%–100%, the insects are susceptible to the insecticide; 80%–97% means additional verification is required, and <80% mortality shows that the insects are resistant. The results of the bioassays on both strains of Ae. aegypti are shown in the Table. The
WHO adult bioassay The bioassay procedure of WHO was used.[9] Sugar-fed, 3–5-days-old adult female mosquitoes were tested. Batches of 25 adults were introduced into pre-holding tubes prior to being exposed to insecticide-impregnated papers in standard WHO test tubes lined with the impregnated papers. Exposed and control tubes of mosquitoes were covered with black cloth during exposure. Equal numbers of control tests were also carried out by exposing mosquitoes to untreated filter paper for one hour. The experiment was replicated four times. All tests were undertaken at 26 °C ± 2 °C and relative humidity of 70%–80% with a photo period of 12-hour darkness and 12-hour light. The mosquitoes were exposed to the
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Table: Susceptibility status of adults of MyRIDL513A and MyWT Ae. aegypti against various insecticides Lethal time, LT (minutes) LT50 (95% C.L.) 177.73a (110.27 – 1109.69) 132.82b (92.02 – 563.33) 78.44a (68.19-184.44) 9.76b (9.24 – 10.29) 27.65a (27.32 – 27.97) 26.05b (25.66 – 26.45) 28.53a (27.94 – 29.15) 127.99b (101.99 – 181.47) 11.75a (11.41-12.08) 14.21b (13.92-14.52) 13.75a (13.32 – 14.18) 13.79b (13.44 – 14.12) 5.12a (4.93 – 5.31) 10.12b (9.88 – 10.36) LT90 (95% C.L.) 493.54a (82.79 – 115.72) 298.23b (152.02 – 4100.77) 104.85a (80.81-524.47) 21.07b (19.18 – 23.64) 32.56a (32.01 – 33.21) 32.21b (31.53 – 33.01) 40.29a (38.91 – 41.97) 810.42b (460.66 – 2016.41) 19.80a (18.97-20.81) 21.39b (20.60-22.30) 23.05a (22.09 – 24.17) 18.50b (17.91 – 19.24) 6.67a (6.33 – 7.17) 12.81b (12.41 – 13.32) 24 hours post-exposure mortality (%) 48a 33b 98.7a 96b 100a 100b 100a 88b 100a 100b 100a 100b 100a 100b
Insecticide
Dosage
Regression line
Susceptibility status
Y = 2.89x – 30.37 Y = 3.69x – 39.72 Y = 10.17x – 116.45 Y = 3.83x – 37.07 Y = 18.05x – 201.56 Y = 13.92 x – 153.97 Y = 8.55x – 92.93 Y = 1.59x – 14.25 Y = 12.57x – 11.11 Y = 13.89x – 11.11 Y = 5.72x – 69.28 Y = 10.03x – 106.77 Y = 11.15x – 114.46 Y = 12.51x – 132.73
Resistant Resistant Susceptible Tolerant Susceptible Susceptible Susceptible Tolerant Susceptible Susceptible Susceptible Susceptible Susceptible Susceptible
DDT
4%
Fenitrothion
1%
Malathion
5%
Propoxur
0.1%
Permethrin
0.75%
Lambdacyhalothrin
0.15%
Cyfluthrin
0.05%
: MyRIDL513A : MyWT (Lab strain) C.L.: Confidence Limit a b
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LT50 value is commonly accepted as the basis for comparison in the investigation of relative toxicities among the insecticides under tests. The results show that both the MyRIDL513A and MyWT strains were resistant to DDT. Resistance to DDT could be due to the genetic origin of the mosquitoes because both possessed a Malaysian genetic background. DDT has been used in the past to control Ae. aegypti, and though its application was stopped in 1957 and substituted by dieldrin,[13] the resistance phenotype has remained in this laboratory strain. Natural pre-existing resistance of Ae. aegypti against DDT has been reported from Malaya[14] (former name for Malaysia), indicating this phenotype is also maintained in the wild. Generally, against the MyRIDL513A strain, the resistance decreased in the order of: DDT > fenitrothion > propoxur > malathion > lambdacyhalothrin > permethrin > cyfluthrin. The strain MyWT, on the other hand, responded in a slightly different pattern, and the resistance decreased in the order: DDT > propoxur > fenitrothion > malathion > lambdacyhalothrin > permethrin > cyfluthrin. There was a variation in response to fenitrothion and propoxur in both the strains, but the resistance pattern of malathion and pyrethroids in the above order remained the same for both the strains. However, exposure of RIDL adults to the discriminating dosages of malathion, permethrin, cyfluthrin and lambdacyhalothrin induced 100% mortality 24 hours posttreatment, indicating that the RIDL mosquito was also susceptible to these insecticides. Strains that are resistant to DDT have been shown to have moderate resistance to pyrethroids, whereas permethrin resistance
resulted in strong resistance to both permethrin and DDT.[15] However, our study indicated that there was no cross-resistance to pyrethroids and this was in accordance with a study in Thailand on wild-type mosquitoes.[16] Fenitrothion and propoxur gave 98.7% and 100% mortality for MyRIDL513A, indicating high susceptibility of this strain. On the other hand, the MyWT strain showed 96% and 88% mortality against fenitrothion and propoxur, indicating possible tolerance of this strain to these insecticides. The development of tolerance could be due to the selection for resistance in the mosquitoes resulting from agricultural application.[17] The MyWT strain, originated from field-caught Ae. aegypti, may have been exposed and selected for resistance against these insecticides. Nevertheless, the trend in the susceptibility status for both the strains was similar. In summary, both the MyRIDL and MyWT strains of Ae. aegypti exhibited almost identical levels of susceptibility/resistance to insecticides. Therefore, the process of outcrossing RIDL to a Malaysian strain has not changed the insecticide-susceptibility status.
Acknowledgement We thank the Director-General of Health, Malaysia, and the Director, Institute for Medical Research (IMR), for permission to publish this paper. This study was supported by a grant (No JPP-IMR-053-07) from the National Institutes of Health, Ministry of Health, Malaysia. Thanks are also due to the staff of the Medical Entomology Unit, IMR, for their assistance and to Oxitec Ltd, U.K., for the provision of the RIDL strain.
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References [1] Skae FM. Dengue fever in Penang . BMJ 1902;2:1581–2. [2] Ministry of Health. Dengue and chikungunya vases 2009; http://www.moh.gov.my/ MohPortal/Index.jsp – accessed 30 June 2010. [3] Brown AWA. Insecticide resistance in mosquitoes: a pragmatic review. J Am Mosq Control Assoc 1986;2:123-40. [4] Alphey L. Re - engineering the sterile insect technique. Insect Biochem Mol Biol 2002;32(10):1243-1247. [5] Alphey L, Benedict M, Bellini R, Clark GG, Dame DA, Service MW, Dobson SL. Sterileinsect methods for control of mosquito-borne diseases: an analysis. Vector Borne Zoonotic Dis 2010;10(3):295-311. [6] Phuc HK, Andreasen MH, Burton RS, Vass C, Epton MJ, Pape G, Fu G, Condon KC, Scaife S, Donnelly CA, Coleman PG, White-Cooper H, Alphey L. Late-acting dominant lethal genetic systems and mosquito control. BMC Biol 2007;5:11. [7] Lee HL, Joko H, Nazni WA, Seshadri Vasan. Comparative life parameters of transgenic and wild strain of Aedes aegypti (L.) in the laboratory. Dengue Bull 2009 (submitted). [8] Nimmo D, Gray P . Mosquito rearing protocol. Intensive Workshop on Wild Type and Genetically Sterile Aedes Mosquitoes, 26 Sept – 2 Oct 2007, Kuala Lumpur Malaysia, pp: 27-34. [9] World Health Organization. Instruction for determining the susceptibility of resistance of adult mosquitoes to organochlorines, organophosphates and carbamate insecticide diagnostic test. World Health Organization Mimeograph 1981; WHO. VBC/81.8067. World Health Organization. Discriminating concentrations of insecticides for adult mosquitoes. 1998; WHO/CDS/CPC/ MAL/98.12. [10] Abbott WS. A method for computing the effectiveness of an insecticide. J Econ Entomol 1925;18:265-7. [11] Raymond M. Log-probit analysis basic programme of microcomputer. Cohiers ORSTOM Serie. Entomology Medicale et Parasitologie 1985;23:117-121. [12] World Health Organization. 10th report of the WHO expert committee on vector biology & control: resistance of vector and reservoir of disease to pesticides. Technical Report Series 737. Geneva: WHO, 1986. [13] Macdonald WW. Resurvey Of Aedes aegypti at Kuala Lumpur Airport. Medical J Malaya 1958;XIII:179-86. [14] Shidrawi GR. Laboratory tests on mosquito tolerance to insecticides and the development of resistance by Aedes aegypti. Bull Wld Hlth Org 1957;17:377-411. [15] Grant DF, Matsumura F. Glutathione S-transferase 1 and 2 in susceptible and insecticide resistant Aedes aegypti. Pestic Biochem Physiol 1989;33:132-43. [16] La-aied Prapanthadara, Nongkran Promtet, Surangchit Koottathep, Pradya Somboon, Wonnapa Suwonkerd, Lynn McCarroll, Janet Hemingway. Mechanisms of DDT and permethrin resistance in Aedes aegypti from Chiang Mai, Thailand. Dengue Bull 2002;26:185-9. [17] Lines JD. Do agricultural insecticides select for insecticide resistance in mosquitoes? A look at the evidence. Parasitology Today 1988;4:S17-S20.
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