Jpn. J. Infect. Dis., 68, 33–37, 2015
Original Article Susceptibility of the Sandfly Phlebotomus argentipes Annandale and Brunetti (Diptera: Psychodidae) to Insecticides in Endemic Areas of Visceral Leishmaniasis in Bihar, India
Ram Singh1* andPramodKumar2 1National Centre for Disease Control, Patna branch, Bihar; and 2National Centre for Disease Control, Delhi, India
SUMMARY: We present the results of susceptibility tests conducted on the sandfly Phlebotomus argen- tipes, the vector of visceral leishmaniasis in India. Adult P. argentipes insects were collected from 42 vil- lages in 6 districts of the state of Bihar, India, as follows: Patna, Vaishali, Muzaffarpur, Samastipur, Sheohar, and Sitamarhi. These adult insects were exposed to 4z DDT-, 5z malathion-, and 0.05z deltamethrin-impregnated papers using a WHO test kit by following the standard procedures. In 16 (38.1z) of 42 villages surveyed, the P. argentipes populations developed resistance to DDT. Susceptibil- ity tests using the organophosphate malathion in 22 villages revealed that in 1 (4.5z) village, the species developed resistance to this insecticide. P. argentipes was, however, highly susceptible to the synthetic pyrethroid deltamethrin. For long-term vector control of P. argentipes, it will be necessary to overcome the threat of insecticide resistance in this species.
susceptibility test in this region (10). DDT resistance in INTRODUCTION P. argentipes populations was reported from some kala- Visceral leishmaniasis (VL) is a major public health azar-endemic areas in Bihar (11,12). However, informa- problem in India. Bangladesh, Nepal, and India signed tion on the status of insecticide resistance in the sandfly a memorandum of understanding and set an elimination is limited. Considering that the development of target of less than 1 case in 10,000 people by 2015 (1,2). resistance was reported in some places after prolonged The disease is transmitted by the bite of infected female use of DDT during IRS in endemic areas of Bihar, the sandflies, (Phlebotomus argentipes Annandale and present study was planned to investigate the susceptibil- Brunetti [Diptera: Psychodidae]), a well-known vector ity of P. argentipes in larger areas. The study was con- of VL called as kala-azar in India (3). P. argentipes is in ducted in highly endemic areas of VL to generate data general zoophilic but is also an opportunistic an- on the pattern of the susceptibility of P. argentipes to thropophile in nature (4). It prefers to breed in the soil DDT. This study also provided an opportunity to identi- of cattle shelters (5). The resting locations of adults are fy a potential insecticide that could be used as an alter- inside the cracks and crevices in the walls of human nate vector control strategy for kala-azar in the state of dwellings and cattle shelters (6), which are the target Bihar. Therefore, the organophosphate malathion and sites for the indoor residual spraying (IRS) of insecti- the synthetic pyrethroid deltamethrin were also tested, cides. Kala-azar was believed to have disappeared from as these are being widely used during IRS for the endemic areas during 1960–1970 as a collateral benefit malaria vector control program in India. of spraying DDT for malaria vector control under the National Malaria Control Program 1953 and National MATERIALS AND METHODS Malaria Eradication Program 1958 (7). It was a com- mon belief that the populations of P. argentipes were The study was conducted in 42 villages in 6 districts of effectively suppressed because of their higher level of Bihar, India. Among these villages, Hulashchak was susceptibility to DDT (8). Withdrawal of DDT spraying selected as the control village, as only one round of after 1962 by the National Malaria Eradication Pro- DDT spraying was performed in 1996 in 30 years. All gram was assumed to have resulted in increased vector the other villages were sprayed with DDT as part of the populations and consequently the resurgence of kala- kala-azar control program in the state of Bihar. The 6 azar during the mid-70s in the state of Bihar in India study districts of the state, Patna (25°25′N, 85°10′E), (8,9). Vaishali (25°30′N, 84°85′E), Muzaffarpur (26°23′N, Prior to 1979, P. argentipes wasconsideredtobe 85°45′E), Samastipur (25°5′N, 85°5′E), Sheohar highly susceptible to the insecticide of DDT in the WHO (26°2′N, 85°03′E), and Sitamarhi (26°49′N, 85°05′E), were selected based on their high endemicity of VL. Adult sandflies were collected from human dwellings Received August 14, 2013. Accepted May 30, 2014. and cattle shelters in each village in the early morning J-STAGE Advance Publication November 25, 2014. hours (0500–0700) with the help of a torch light and an DOI: 10.7883/yoken.JJID.2013.262 aspirator tube between March 2004 and November *Corresponding author: Mailing address: National Centre 2007. A total of 42 villages were evaluated for suscepti- for Disease Control, Patna branch, Bihar, India. Tel and bility to DDT, and because of the nonavailability of Fax: ＋916122572849, E-mail: ramsingh48＠hotmail.com sufficient numbers of sandflies for testing, malathion
33 Table 1. Susceptibility status of P. argentipes to 4z DDT in the WHO tube test
4z DDT at 1 h exposure Control
District Village No. exposed z mortality No. exposed z mortality Corrected mortality Result1) Patna Hulashchak 90 100 30 0 100 S Mahinama Bazar 60 100 20 0 100 S Shanti nagar 60 96.7 15 0 96.7 T Janipur 60 100 15 0 100 S Dariyapur 60 100 20 0 100 S Gulmahia 60 66.7 18 0 66.7 R Gulmahia bagh 86 74.4 20 0 74.4 R Poonadih 60 90 16 0 90 T Naya Tola 60 80 20 0 80 T Sarayia 8095300 95 T Bakhara Chakia 45 95.5 15 0 95.6 T BhagahaTola 60 100 20 0 100 S Akopur 60 100 20 0 100 S Pulia Tola 60 100 20 5 100 S Majhaulia 52 98.1 20 0 98.1 S Vaishali Daulatpur Chandi 45 77.8 25 8 75.9 R MilePakari 60 53.3 15 0 53.3 R Rahimapur Pethia 60 78.3 15 6.7 76.7 R Kuwari bari Tola 48 68.8 16 6.3 66.5 R Muneem Chak 60 70 20 0 70 R Kanchanpur 60 95 15 0 95 T Jamalpur 60 93.3 20 0 93.3 T Wazitpur 48 97.9 20 0 97.9 T Kuwaribari Tola 60 73.3 18 0 73.3 R Islampur 60 63.3 25 8 60.1 R Chainpuramile pakari 60 85 18 5.6 84.1 T Muzaffarpur Gopnathpur Dokra 45 84.4 15 0 84.4 T GhandigramThuhma 50 78 15 6.7 76.4 R Chandra Hati 60 83.3 15 6.7 82.1 T Vijay Chhapara 45 93.3 16 6.3 92.9 T Pana pur 45 95.5 20 10 95 T Tarma Dushal Tola 45 95.6 18 0 95.6 T Akhatiyarpur 45 91.1 16 0 91.1 T Motipur 80 100 30 0 100 S Makshodpur 80 100 30 0 100 S Samastipur Jhhaypura 60 43.3 15 0 43.3 R Chandchouh 60 48.3 15 0 48.3 R Gangapur 55 54.5 15 0 54.5 R Bangra 50 64 15 6.7 61.4 R Mubarkpur 45 55.6 15 0 55.6 R Sheohar Rasheedpur 60 98.3 20 0 98.3 S Sitamarhi Ganeshpur 70 72.9 15 0 72.9 R
1): S, susceptible; T, tolerance; R, resistance. and deltamethrin susceptibility tests were performed sliding unit. The exposure tubes marked with red dots only in 22 and 20 villages, respectively. were lined with insecticide-impregnated paper and 1 The collected sandflies were brought to the laboratory control with carrier oil. All P. argentipes specimens in and released into a cage for acclimatization for 1 h ex- the holding tubes were transferred to the exposure tube clude insects that died of transportation stress. P. argen- using the sliding unit, and insects were kept in the ex- tipes specimens that were retained were used for suscep- posure tube for 1 h. The specimens were then trans- tibility tests, whereas other species of sandflies were ferred to a holding tube containing a 10z glucose removed. Papers impregnated with 4z DDT, 5z solution-soaked cotton pad. Mortality was recorded malathion, and 0.05z deltamethrin were used in sus- after 24 h of exposure. Abbot's formula was used to ceptibility tests by following the standard WHO suscep- correct the mortality rate if the mortality rate in the con- tibility test methods (13,14). trol was 5–20z ([(ztest mortality － zcontrol mortali- Blood-fed and active half-gravid females of P. argen- ty) × 100]/[100 － zcontrol mortality]). The tempera- tipes were used for the susceptibility test. Batches of ture and relative humidity were maintained at 25 ± 29C 15–25 sandflies were put in each holding tube with the and 75–80z, respectively. Three or 4 replicates were help of an aspirator tube through the filling hole in the performed for each insecticide in each village. The sus-
34 Insecticides Resistance in Sandfly P. argentipes
Table 2. Susceptibility status of P. argentipes to 5z malathion in the WHO tube test
5z malathion at 1 h exposure Control
District Village No. exposed z mortality No. exposed z mortality Corrected mortality Result1) Patna Dariyapur 45 100 20 0 100 S Gulmahiya 45 100 20 0 100 S Gulmahiya bagh 45 100 15 0 100 S Poonadih 45 100 20 0 100 S Bakharachakiya 45 100 15 0 100 S Akopur 45 100 16 0 100 S Vaishali Rahimapur Pethia 60 96.7 20 10 96.7 T Bidupur 45 100 16 0 100 S Muneemchak 45 100 18 0 100 S Muzaffarpur Gopnathpur Dokra 45 95.6 16 0 95.6 T Gandhigram Thumha 45 100 15 0 100 S Chandrahati 60 100 20 0 100 S Pana pur 45 100 15 0 100 S Tarma Dushal Tola 45 100 15 0 100 S Akhatiyarpur 45 100 15 6.7 100 S Vijayee Chhapara 45 100 15 0 100 S Samastipur Jhhaipura 45 84.4 15 0 84.4 T Chandchouh 40 87.5 15 0 87.5 T Gangapur 50 74 15 0 74 R Bangra 45 88.9 18 0 88.9 T Mubarakpur 45 95.6 16 0 95.6 T Sarayia 80 100 20 5 100 S
1): see Table 1 footnote.
Table 3. Susceptibility status of P. argentipes to 0.05z deltamethrin in the WHO tube test
0.05z deltamethrin Control at 1 h exposure
District Village No. exposed z mortality No. exposed z mortality Corrected mortality Result1) Patna Hulashchak 45 100 15 0 100 S Mahinam bazaar 45 100 15 0 100 S Shanti nagar 45 100 16 0 100 S Janipur 45 100 15 0 100 S Dariyapur 45 100 15 0 100 S Nayatola 45 100 15 0 100 S Sarayia 45 100 15 0 100 S Akopur 45 100 15 0 100 S B. danapur 45 100 15 0 100 S Vaishali Muneem Chak 45 100 15 0 100 S Kanchanpur 45 100 15 0 100 S Jamalpur 45 100 15 0 100 S Wazitpur 45 100 15 0 100 S Kanchanpur 45 100 15 0 100 S Kuwaribari Tola 50 100 15 0 100 S Muzaffarpur Vijay Chhapara 45 100 15 13.3 100 S Gandhigram Thumba 45 100 17 0 100 S Pana pur 45 100 18 0 100 S Tarma Dushal Tola 45 100 16 6.3 100 S Akhatiyarpur 45 100 15 0 100 S
1): see Table 1 footnote. ceptibility results were interpreted as per the WHO for spermetheca and pharyngeal teeth following the recommendation for malaria vectors and the guidelines guidelines prescribed for sandfly identification (15). In- of Dinesh et al. (2010) for the sandfly as follows: mor- secticide-impregnated and control papers along with a tality of 98–100z indicated susceptible; 80–97z indi- WHO test kit were obtained from National Vector cated tolerance; and ＜80z indicated resistance (13,14). Borne Disease Control Program, Government of India, To confirm the identity of the evaluated sandflies, all Delhi. dead and living sandflies were dissected and observed
35 which reduced the duration of the effectiveness of DDT RESULTS spraying. The natural phenomenon of the development The results of the susceptibility test performed on of resistance/tolerance on the prolonged use of a highly field populations of P. argentipes with different insec- persistent organochlorine such as DDT also occurred in ticide-impregnated papers are shown in Tables 1–3. P. argentipes. The susceptibility test on the P. argentipes popula- The present study is the first large study to collect tions with DDT-impregnated papers conducted in 42 data from 42 villages. In addition, the present study is villages revealed that the populations in 11 villages also the first to attempt an assessment of the develop- (26.2z) were susceptible, whereas tolerance and ment of resistance in P. argentipes in larger endemic resistance were observed in 15 (35.7z) and 16 villages areasofVL,with1areaexhibitinghighlevelsofresist- (38.1z), respectively (Table 1). The mortality rate ob- ance to DDT, as indicated by a mortality rate of 43.3z. served during the test was ＜60z in 31.2z of villages In India, malathion is widely used during IRS in the and 60–80z in 26.2z of villages. The population in the malaria vector control program as well as in agriculture. unsprayed control village, Hulashchak, was found to be The populations of P. argentipes werefoundtohave 100z susceptible to DDT (Table 1). high susceptibility to malathion in Bahpur of Patna dis- Tests of the susceptibility of P. argentipes popula- trict in 2001 (17), whereas the present study recorded a tions to malathion conducted in 22 villages revealed that lower mortality rate of 74z for P. argentipes in Gan- the populations in 15 villages (68.2z) were 100z sus- gapur of Samastipur district. The mortality rate for P. ceptible, whereas tolerance and resistance were observed argentipes populations exposed to malathion in 6 of 24 in 6 (27.3z) and 1 village (4.5z), respectively (Table 2). villages evaluated for malathion susceptibility ranged A mortality rate of 100z for malathion was observed in from 84.4 to 96.7z, suggesting the development of 68.2z of villages, whereas mortality rates of 84.4–96.7 resistance/tolerance to this insecticide in P. argentipes and 74z were observed in 27.3 and 4.5z of villages, re- populations in endemic areas of kala-azar. In nonen- spectively. demic areas, P. argentipes populations also displayed Meanwhile, susceptibility testing revealed 100z sus- resistance to this insecticide (21). P. argentipes speci- ceptibility to deltamethrin among P. argentipes popula- mens were fully susceptible to the synthetic pyrethroid tions in 20 villages (Table 3). deltamethrin in the study areas (17,19,20). In conclusion, populations of P. argentipes,thevec- tor of VL, exhibited a high level of resistance/tolerance DISCUSSION to DDT in VL-endemic areas of the state of Bihar, In- Insecticides play an important role in reducing the dia. Interestingly, resistance/tolerance to malathion disease burden as an effective vector control measure. also developed in larger areas. The sandfly was suscepti- The current vector control programs for VL rely heavily ble to deltamethrin in all of the study areas in Bihar. on IRS of DDT at 1 g/m2 in human dwellings as well as WHO has recommended the rotation of insecticides to in cattle shelters to control the sandfly vector P. argen- prevent the spread of insecticide resistance. Insecticides tipes (16). The present study illustrated the susceptibility with different modes of action should be used alternate- of P. argentipes to 4z DDT, 5z malathion, and 0.05z ly in rotation (24). Therefore, it is recommended that deltamethrin. Among 42 villages assessed for suscepti- deltamethrin should be used to which the P. argentipes bility to DDT, resistance was observed in 38.1z of vil- species is highly susceptible in the VL-endemic area in lages, in which the mortality rates varied between 43.3 Bihar. This will be in the larger public interest to meet and 76.7z. Various workers have reported resistance to the target guidelines set by the government of India to DDT among P. argentipes populations in endemic areas eliminate VL from this region. This study provided an of Bihar, with different levels of mortality. In 1989, 1 opportunity for Indian policy makers to realize the village of Samastipur district was found to have a mor- potential hazards of using a single insecticide such as tality rate as low as 40z (11), whereas in Ravidas Tola DDT for many decades and implement measures to of Vaishali district (1999–2000), the mortality rate for combat resistance through an alternate potential insecti- P. argentipes following exposure to DDT was found to cide such as deltamethrin. be 71z (12). In 2001, in A. Kawahi and Singharai of Vaishali, the mortality rates for P. argentipes following Conflict of interest None to declare. exposure to DDT were 52.4z and 33.3z, respectively (17). In Kanhara Hardas of the Muzaffarpur distinct and Shing Rai Kawahi of the Vaishali district, the mor- REFERENCES tality rates for P. argentipes following exposure to DDT 1. WHO Regional Office for South-East Asia. Regional strategic were reported to be as 56.5 and 54.7z, respectively framework for elimination of kala-azar from the South-East Asia Region (2011–2015). SEA-CD-239. 2012. (18). Similar findings were reported for the development 2. Gupta A, Nagar M, Mishra SS, et al. Visceral leishmaniasis (kala- of resistance in P. argentipes in endemic areas for VL in azar) elimination from Indian Sub-continent by 2015? Int J Trop India (19,20). P. argentipes specimens from nonendem- Dis Health. 2013;3:73-81. ic areas also developed tolerance to DDT (21). In 1996, 3. Swaminath CS, Shortt HE, Anderson LA. Transmission of Indi- an kala-azar to man by the bite of Phlebotomus argentipes. ann. P. argentipes populations reappeared in DDT-sprayed and brun. 1942. Indian J Med Res. 1942;30:473-7. houses 2.5 months after treatment in Vaishali district 4. Mukhopadhyay AK, Chakravarty AK. Bloodmeal preference of (22), whereas these insects previously (1982–83) reap- Phlebotomus argentipes & Ph. papatasi of north Bihar, India. In- peared after 6 months in the kala-azar-endemic areas of dian J Med Res. 1987;86:475-80. Darbhanga district of Bihar (23). These findings indi- 5. Singh R, Lal S, Saxena VK. Breeding ecology of visceral leishmaniasis vector sandfly in Bihar state of India. Acta Trop. cate the development of tolerance in vector species,
3636 Insecticides Resistance in Sandfly P. argentipes
2008;107:117-20. 16. National Vector Borne Disease Control Programme, Government 6. Saxena VK. Sandflies from a kala-azar outbreak area of district of India, Guidelines on vector control in KA elimination, 1-21. Darbhanga (Bihar), India. J Commun Dis. 1987;19:405-7. Available at
3737