Resistance to niclosamide in Oncomelania hupensis ...

1

Resistance to niclosamide in Oncomelania hupensis, the intermediate host of Schistosoma japonicum: should we be worried? JIAN-RONG DAI 1,2,3 , YOU-ZI LI 1,2,3 , WEI WANG 1,2,3 *, YUN-TIAN XING 1,2,3 , GUO-LI QU 1,2,3 and YOU-SHENG LIANG 1,2,3 * 1

Jiangsu Institute of Parasitic Diseases, 117 Yangxiang, Meiyuan, Wuxi City, Jiangsu Province, 214064, People’s Republic of China 2 Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, 117 Yangxiang, Meiyuan, Wuxi City, Jiangsu Province, 214064, People’s Republic of China 3 Jiangsu Provincial Key Laboratory of Molecular Biology of Parasites, 117 Yangxiang, Meiyuan, Wuxi City, Jiangsu Province, 214064, People’s Republic of China (Received 26 February 2014; revised 10 April and 2 May 2014; accepted 3 May 2014) SUMMARY

As the currently only available molluscicide, niclosamide has been widely used for snail control for over 2 decades in China. There is therefore a concern about the emergence of niclosamide-resistant snail populations following repeated, extensive use of the chemical. The purpose of this study was to investigate the likelihood of niclosamide resistance in Oncomelania hupensis in China. Active adult O. hupensis snails derived from 20 counties of 10 schistosomiasis-endemic provinces of China, of 10 snails in each drug concentration, were immersed in solutions of 1, 0·5, 0·25, 0·125, 0·063, 0·032, 0·016 and 0·008 mg L− 1 of a 50% wettable powder of niclosamide ethanolamine salt (WPN) for 24 and 48 h at 25 °C, and the median lethal concentration (LC50) was estimated. Then, the 24- and 48-h WPN LC50 values were compared with those determined in the same sampling sites in 2002. The results indicated that the 24- and 48-h WPN LC50 values for O. hupensis were not significantly different from those determined in 2002 (P = 0·202 and 0·796, respectively). It is concluded that the current sensitivity of O. hupensis to niclosamide has not changed after more than 2 decades of repeated, extensive application in the main endemic foci of China, and there is no evidence of resistance to niclosamide detected in O. hupensis. Key words: Schistosoma japonicum, Oncomelania hupensis, niclosamide, molluscicidal activity, resistance, China.

INTRODUCTION

Schistosomiasis is a snail-borne neglected parasitic disease which affects over 207 million people in 76 countries, with a further 779 million people at risk of infection (Steinmann et al. 2006). Schistosomiasis japonica, caused by the infection of the blood fluke Schistosoma japonicum (Platyhelminthes: Trematoda), is mainly endemic in the People’s Republic of China, the Philippines and parts of Indonesia (Engels et al. 2002; Zhou et al. 2010). In China, the disease was historically endemic in 12 southern provinces (Chen and Feng, 1999). The control efforts since the 1950s have achieved a remarkable reduction in the prevalence of the infection and the disease burden among humans (Engels et al. 2005; Utzinger et al. 2005; Zhou et al. 2005), and historically, the strategy to control the transmission of S. japonicum in China has shifted three times: disease elimination strategy through * Corresponding authors: Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, 117 Yangxiang, Meiyuan, Wuxi City, Jiangsu Province, 214064, People’s Republic of China. E-mail: [email protected] or [email protected] Parasitology, Page 1 of 9. © Cambridge University Press 2014 doi:10.1017/S0031182014000870

snail control (1950s to early 1980s), morbidity control strategy based on chemotherapy (mid 1980s to 2003) and integrated control strategy (since 2004) (Collins et al. 2012). However, schistosomiasis japonica remains a current major public-health concern in China, which is one of the highest priorities in communicable disease control defined by the central government (Zhou et al. 2005). Currently, about 0·7 million people living in China are thought to have this disease (Zhou X. N. et al. 2007), and the intermediate host Oncomelania hupensis (Gredler, 1881) is still present in numerous areas covering a total of 3·73 billion m2 (Zheng et al. 2012). As the transmission of this neglected tropical disease is governed by the geographic distribution of O. hupensis, control of this snail, as a major part of the National Schistosomiasis Control Program of China, has been implemented in attempts to interrupt the transmission of the disease (Zhou, 2005; Wang et al. 2008, 2009; McManus et al. 2009). Many approaches have been used in attempt to control the intermediate host snails in China, including environmental modification, physical methods, tree building, molluscicide treatment and biological control (Yuan et al. 2005), in which

Jian-Rong Dai and others

2

Table 1. Location and environmental types of the snail sampling sites in China Snail sampling site

Environmental type

Code East longitude (E)

North latitude (N)

Yongsheng County, Yunnan Province Renshou County, Sichuan Province Xichang County, Sichuan Province Lushan County, Sichuan Province Hanshou County, Hunan Province Junshan County, Hunan Province Yushan County, Jiangxi Province Anqing County, Anhui Province Guichi County, Anhui Province Guangde County, Anhui Province Tongling County, Anhui Province Hanjiang County, Jiangsu Province Jurong County, Jiangsu Province Changshan County, Zhejiang Province Jingshan County, Shanghai City Songjiang County, Shanghai City Donghan County, Fujian Province Qianjiang County, Hubei Province Xingguo County, Hubei Province Ezhou County, Hubei Province

Hill Hill Hill Hill Lake and marshland Lake and marshland Hill Lake and marshland Lake and marshland Hill Lake and marshland Lake and marshland Hill Hill Plain with waterway networks Plain with waterway networks Hill Lake and marshland Lake and marshland Lake and marshland

A B C D E F G H I J K L M N O P Q R S T

26·45° 30·08° 27·89° 30·13° 28·48° 29·35° 28·56° 30·49° 30·61° 30·99° 30·93° 32·17° 31·58° 28·55° 30·52° 30·55° 25·33° 30·17° 29·51° 30·25°

molluscicide treatment is the most widely used method for snail control due to wide application coverage, easy procedure and fast action (Yang et al. 2012). Since the 1950s, many chemicals have been screened and evaluated for molluscicidal actions in the laboratory, and some agents have been used for snail control in the schistosomiasis-endemic field of China, including sodium pentachlorophenol, calcium cyanamide, metaldehyde, niclosamide and META-Li (Zhu et al. 1999, 2006; Zhou et al. 2002; Wu et al. 2006). However, most of these chemicals are no longer used due to high cost, a comparatively low efficacy and heavy environmental pollution (Zhou, 2005). Currently only one chemical molluscicide, niclosamide, is acceptable for operational use in snail control programmes (WHO, 1992); however, the agent suffers from the problem of being difficult to dissolve in both water and organic solvents. Since 1992, when the World Bank Loan Project for Schistosomiasis Control was initiated in China, a novel formulation of niclosamide, a 50% wettable powder of niclosamide ethanolamine salt (WPN) was introduced and has replaced other molluscicides to become the only molluscicide used for snail control in the endemic field of China (Xianyi et al. 2005). WPN is recommended by the Ministry of Health, the People’s Republic of China at a dose of 1 mg L− 1 for snail control in the field (MOH, 2000). Following extensive, long-term, repeated use for more than 2 decades, the likelihood of emergence of niclosamide resistance in the intermediate host snails has received much attention (Cao et al. 2012). Therefore, a systematic survey of the sensitivity to niclosamide in O. hupensis is of great significance to assess the molluscicidal actions of niclosamide in the endemic field and monitor the emergence of niclosamideresistant snail populations. This study was therefore

100·45° 104·01° 102·28° 102·91° 112·02° 112·05° 118·06° 117·18° 117·41° 119·55° 117·82° 119·24° 119·12° 118·30° 121·15° 121·04° 119·22° 112·41° 115·21° 114·51°

designed, in the laboratory, to evaluate the molluscicidal activities of WPN against O. hupensis sampled from 20 counties of 10 schistosomiasis-endemic provinces in China in 2012 and compare them with those determined in the same sampling sites in 2002, so as to investigate the likelihood of resistance to niclosamide in O. hupensis snails. METHODS

Study area and snail populations Adult O. hupensis snails were sampled by individual picking with forceps (Mao, 1990), from snail habitats in 20 counties of 10 schistosomiasis-endemic provinces of China during the period from 7 April through 7 May 2012 (Table 1, Fig. 1). After feeding in the laboratory for 24 h, active adult snails with 7–8 spirals were randomly grouped for the evaluation of molluscicidal activity in the laboratory. Drug solutions WPN was kindly provided by the Nanjing Essence Fine Chemical Co., Ltd (Nanjing, China; lot number: 1012031). WPN was weighed and solutions of 1, 0·5, 0·25, 0·125, 0·063, 0·032, 0·016 and 0·008 mg L− 1 of niclosamide were prepared with dechlorinated tap water for the subsequent experiments. Laboratory evaluation of molluscicidal activity One hundred-millilitre flasks were filled with the solutions, 10 active adult snails transferred to each and the flasks covered with gauze to prevent their escape. Snails in flasks of dechlorinated water without

Niclosamide resistance in O. hupensis

3

Fig. 1. The sampling sites of Oncomelania hupensis snails in China. The O. hupensis snails used for molluscicidal tests were collected from 20 counties of 10 provinces in China, including Yongsheng County of Yunnan Province, Renshou, Xichang and Lushan counties of Sichuan Province, Hanshou and Junshan counties of Hunan Province, Yushan County of Jiangxi Province, Anqing, Guichi, Guangde and Tongling counties of Anhui Province, Qianjiang, Xingguo and Ezhou counties of Hubei Province, Hanjiang and Jurong counties of Jiangsu Province, Changshan County of Zhejiang Province, Jingshan and Songjiang counties of Shanghai City and Donghan County of Fujian Province.

drug treatment served as controls. After being immersed for 24 and 48 h at 25 °C, the snails were washed with dechlorinated water and fed for a further 48 h. Those suspected of being dead were tested by the knocking method (Gönnert, 1961; Webbe, 1961), and the snail mortality was estimated. All tests were performed in triplicate, and the median lethal concentration (LC50) was calculated. Comparison of molluscicidal activity of WPN to that determined in 2002 In April and May 2002, O. hupensis snails were collected from 37 sampling sites in 33 counties of 10 Chinese provinces using a stratified random sampling method, to evaluate their response to WPN. After 24-h feeding in the laboratory, active adult snails, with 10 snails in each drug concentration, were immersed in WPN at concentrations of 1, 0·5, 0·25, 0·125, 0·063, 0·032, 0·016 and 0·008 mg L− 1 for 24 and 48 h at 25 °C, and the molluscicidal tests were performed in triplicate. The LC50 values were then calculated (Dai et al. 2002a). To test the concern about reduced sensitivity of O. hupensis to niclosamide, the LC50 values of WPN for O. hupensis snails estimated in this study were compared with those determined in the 20 same sampling sites in 2002. Ethics statement The study protocol was approved by the Scientific Steering Committee and Ethics Review Committee of Yunnan Institute of Endemic Diseases Control (permission number: 2012-01), Sichuan Provincial Centre for Disease Control and Prevention (permission number: ERC2012-11), Hunan Provincial

Institute of Parasitic Diseases (permission number: 2012-01), Zhejiang Academy of Medical Sciences (permission number: SSERC2012-20), Jiangxi Provincial Institute of Parasitic Diseases (permission number: 2012-05), Anhui Provincial Institute of Parasitic Diseases (permission number: 2012-01), Jiangsu Institute of Parasitic Diseases (permission number: [2012]05), Shanghai Municipal Centre for Disease Control and Prevention (permission number: ERC2011-57), Fujian Provincial Centre for Disease Control and Prevention (permission number: 2011-127) and Hubei Provincial Centre for Disease Control and Prevention (permission number: [2012]-05). All field surveys did not involve endangered or protected species.

Statistical analysis All data were double-entered into a Microsoft Excel database version 2007 (Microsoft Corporation; Redmond, WA, USA) and all statistical analyses were performed using the statistical software SPSS version 17.0 (SPSS Inc.; Chicago, IL, USA). The difference in the sensitivity to WPN in the O. hupensis snails sampled from various sites was tested for statistical significance with analysis of variance (ANOVA), while comparison of the WPN LC50 values for O. hupensis between 2002 and 2012 was performed with Student’s t-test. A P value 0·05). Nearly all snails were dead following immersion in 0·5 (except the snails sampled from Junshan of Hunan province) and 1 mg L− 1 WPN for 48 h, whereas all snails were alive following immersion with 0·008 mg L− 1 WPN for 48 h. Immersion in 0·25, 0·125, 0·063, 0·032 and 0·016 mg L− 1 WPN for 48 h resulted in 86·67–100%, 10–100%, 0–63·33%, 0–20% and 0–10% mortalities of snails, respectively (Table 3). The 48-h WPN LC50 values for the O. hupensis snails derived from 20 sampling sites in China ranged from 0·0442 to 0·1768 mg L− 1, and ANOVA revealed no regional variation (P > 0·05).

Difference in niclosamide LC50 values between 2002 and 2012 There was a minor variation in the 24-h LC50 values of WPN for the O. hupensis snails derived from the same 20 sampling sites estimated between 2012 and 2002 (Fig. 2); however, no significant difference was observed (P = 0·202). Similarly, the 48-h WPN LC50 values varied in the O. hupensis snails between 2012 and 2002 (Fig. 3); however, no significant difference was detected (P = 0·796).

DISCUSSION

Oncomelania hupensis (Gredler, 1881) is the only intermediate host of S. japonicum, which causes schistosomiasis endemic in the Far East, and especially in mainland China. Epidemiological studies have shown that O. hupensis largely determines the geographical range of the parasite (Zhou, 2005). The snail O. hupensis can be separated phenotypically into a ribbed-shelled morphotype, with its distribution restricted to the marshland and lake region and plain region along the middle and lower reaches of the Yangtze River, and smooth-shelled morphotypes which are only detected in mountainous regions (Li et al. 2009; Zhao et al. 2010). The snails from different regions are found to vary in morphology, genetic, physiological and biochemical characteristics, and various subspecies have been differentiated (Davis et al. 1995; Wilke et al. 2000; Zhou Y. B. et al. 2007; Li et al. 2009; Zhao et al. 2010). In addition, it is reported that niclosamide treatment results in varied molluscicidal activities in different endemic

4

foci (Yang et al. 2012). It is still unknown whether such a variation is attributable to the snail populations or reduced niclosamide susceptibility following repeated, extensive use of the agent for more than 2 decades in China. Therefore, there is an urgent need to assess the current molluscicidal activities of niclosamide against O. hupensis snails in regions where since it is the only commercially available chemical it is widely used, and test the concern about the resistance to niclosamide in O. hupensis. Our findings showed that no field-derived O. hupensis snails survived the 24- or 48-h treatment with 1 mg L− 1 WPN, the currently recommended dose for snail control in the endemic field, and nearly all snails (except the snails sampled from Junshan of Hunan province) were dead following immersion in 0·5 mg L− 1 WPN for 48 h. The results from this study demonstrate that the molluscicidal activity of niclosamide against O. hupensis is still high in the main endemic areas of China. In addition, the 24-h WPN LC50 values for the O. hupensis snails derived from 20 sampling sites in China ranged from 0·0981 to 0·2618 mg L− 1, and the 48-h LC50 values between 0·0442 and 0·1768 mg L− 1. Although the LC50 value of WPN varied in the O. hupensis snails from different regions, no regional variation was detected (both P values >0·05), revealed by ANOVA. Compared with the WPN LC50 values determined in the same 20 sampling sites in 2002, no significant differences were found in the 24- and 48-h LC50 values of WPN for O. hupensis snails (P = 0·202 and 0·796). This finding further demonstrates no reduced sensitivity to niclosamide in O. hupensis snails detected in mainland China. Many factors have been reported to affect the efficacy of molluscicidal treatment in the endemic field, including temperature, sunlight, snail habitats, soil content, operational skills, as well as the brand, quality and concentration of the chemical (Paulini, 1958; Oliveira-Filho et al. 1999; Zhou, 2005). In laboratory tests, the temperature, molluscicide solution volume and the duration of snail breeding in the laboratory are found to affect the molluscicidal actions of niclosamide against O. hupensis snails (Zhang et al. 1999; Dai et al. 2002b, 2003). A study to evaluate the sensitivity to niclosamide of O. hupensis snails sampled through a year showed no significant differences in the 24- and 48-h WPN LC50 values for the snails sampled at various months, with minor fluctuations observed (Li et al. 2012). In addition, 10 snails in each drug concentration are considered to be an optimum quantity for molluscicidal tests in the laboratory (Dai et al. 2002c). In the present study, the snails were collected within a month to reduce the effect on the molluscicidal test, and 10 snails were assigned to each dose group for evaluating the molluscicidal actions. Our findings showed that the sensitivity to

Mortality of snails in different concentrations of WPN (%) Snail population

1 mg L− 1

0·5 mg L− 1

0·25 mg L− 1

0·125 mg L− 1

0·063 mg L− 1

0·032 mg L− 1

0·016 mg L− 1

0·008 mg L− 1

LC50 (mg L− 1)

Yongsheng Renshou Xichang Lushan Hanshou Junshan Qianjiang Xingguo Ezhou Yushan Anqing Guichi Guangde Tongling Hanjiang Jurong Changshan Jinshan Songjiang Donghan

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

100 100 96·67 96·67 100 90 100 100 100 100 96·67 100 100 100 100 100 100 100 100 100

90 63·33 80 80 66·67 66·67 100 100 93·33 83·33 86·67 90 60 100 100 40 96·67 100 100 70

66·67 10 0 20 46·67 13·33 33·33 60 20 6·67 40 25 30 80 20 3·33 33·33 50 36·67 5

10 6·67 0 10 10 3·33 0 16·67 6·67 6·67 0 0 0 5 3·33 3·33 10 23·33 6·67 0

0 3·33 0 3·33 0 0 0 0 3·33 0 0 0 0 0 0 0 3·33 3·33 3·33 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0·1115 0·1982 0·2052 0·1627 0·1506 0·2132 0·1402 0·1040 0·1534 0·1250 0·1508 0·1593 0·1895 0·0981 0·1504 0·2618 0·1309 0·1063 0·1308 0·2102


Table 2. Mortality rates and LC50 values of Oncomelania hupensis snails sampled from different regions of China following immersion in various concentrations of WPN for 24 h

5


Table 3. Mortality rates and LC50 values of Oncomelania hupensis snails sampled from different regions of China following immersion in various concentrations of WPN for 48 h Mortality of snails in different concentrations of WPN (%) Snail population

1 mg L− 1

0·5 mg L− 1

0·25 mg L− 1

0·125 mg L− 1

0·063 mg L− 1

0·032 mg L− 1

0·016 mg L− 1

0·008 mg L− 1

LC50 (mg L− 1)

Yongsheng Renshou Xichang Lushan Hanshou Junshan Qianjiang Xingguo Ezhou Yushan Anqing Guichi Guangde Tongling Hanjiang Jurong Changshan Jinshan Songjiang Donghan

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

100 100 100 100 100 93·33 100 100 100 100 100 100 100 100 100 100 100 100 100 100

100 96·67 100 100 100 90 96·67 100 100 100 100 100 100 100 100 86·67 100 100 100 100

93·33 23·33 16·67 33·33 53·33 56·67 50 73·33 100 60 66·67 25 10 100 46·67 13·33 50 80 60 40

20 3·33 0 10 10 6·67 3·33 46·67 63·33 6·67 23·33 5 10 70 0 0 3·33 23·33 23·33 10

0 3·33 0 3·33 0 3·33 0 6·67 0 0 0 0 3·33 20 0 0 10 10 0 10

0 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 3·33 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0·0806 0·1471 0·1577 0·1279 0·1140 0·1152 0·1250 0·0735 0·0570 0·1166 0·0947 0·1436 0·1612 0·0442 0·1279 0·1768 0·0926 0·0788 0·0992 0·1166

6


7

Fig. 2. Comparison of 24-h WPN LC50 values for O. hupensis between 2002 and 2012. The 24-h WPN LC50 values for O. hupensis in all 20 sampling sites in 2012 were compared with those determined in 2002, and no significant difference was detected (P = 0·202), revealed by Student’s t-test.

Fig. 3. Comparison of 48-h WPN LC50 values for O. hupensis between 2002 and 2012. The 48-h WPN LC50 values for O. hupensis in all 20 sampling sites in 2012 were compared with those estimated in 2002, and no significant difference was observed (P = 0·796), detected by Student’s t-test.

niclosamide varied in snail populations sampled from 20 sites of China; however, no regional variation was detected in the 24- and 48-h WPN LC50 values for

O. hupensis snails, which was consistent with previous studies (Dai et al. 2002a, 2014). Further studies are required to investigate the mechanisms


underlying the wide fluctuation in the LC50 values of WPN for O. hupensis snails. The present study shows that the current sensitivity of niclosamide against O. hupensis has not changed after more than 2 decades of repeated, extensive use in China. This is important information for both the public health workers and health policymakers in the field of schistosomiasis control, considering that niclosamide, currently the only chemical available for snail control, plays an essential role in the current Chinese National Schistosomiasis Control Program of China. Currently, a unique WPN regimen (spraying at a dose of 1 mg L− 1) is recommended for field snail control in China (MOH, 2000). It is therefore suggested that the dose of molluscicides should be appropriately adjusted based on the actual field conditions, so as to maximize the efficacy of molluscicidal treatment, and simultaneously reduce resource consumption and environmental pollution. In conclusion, the current efficacy of niclosamide against O. hupensis snails appears satisfactory in China, and no evidence of niclosamide resistance in O. hupensis is detected in China. However, it does not mean that resistance cannot develop or that the response of O. hupensis snails to niclosamide has no regional variation. We, therefore, should not reduce our vigilance to the possible emergence of niclosamide resistance in the intermediate host snails. A dynamic monitoring of the sensitivity to niclosamide in O. hupensis seems justified, which would be of great significance for the prevention, reduction and active management of niclosamide-resistant snail populations, as well as for the elimination of schistosomiasis japonica in China.

FINANCIAL SUPPORT

This work was supported by the National Important Sci-Tech Special Projects [2012ZX10004-220], the Natural Science Foundation of China [81071379], the Jiangsu Province’s Outstanding Medical Academic Leader Programme [LJ201132], Jiangsu Department of Health [X201103] and Jiangsu Government Scholarship for Overseas Studies [JS-2012-130].

CONFLICTS OF INTEREST

The authors have declared that no competing interests exist.

AUTHOR CONTRIBUTIONS

JRD, WW and YSL conceived and designed the study; JRD, YZL, WW, YTX and GLQ conducted the study. JRD and YZL collected the data and performed analysis of data. WW prepared the first draft of the manuscript; YSL provided strategic advice and assisted with editing of the manuscript. All authors read and approved the final version of the manuscript.

8 REFERENCES Cao, Z. G., Wang, T. P., Zhang, S. Q., Tian, X. G., Zhu, L., Zhang, L. S., Yao, G. X., Jin, W. and Yang, W. P. (2012). Experimental study on the resistance of Oncomelania snails to niclosamide. Journal of Pathogen Biology 7, 352–353, 376. Chen, M. G. and Feng, Z. (1999). Schistosomiasis control in China. Parasitology International 48, 11–19. Collins, C., Xu, J. and Tang, S. (2012). Schistosomiasis control and the health system in P.R. China. Infectious Diseases of Poverty 1, 8. Dai, J. R., Zhou, X. N., Liang, Y. S., Zhang, Y. P., Jiang, Y. J., Xi, W. P., Huang, Y. X., Chen, C., Huang, M. X. and Zhu, Y. C. (2002a). Sensitivity of Oncomelania snail to niclosamide in China. Chinese Journal of Parasitology and Parasitic Diseases 20, 101–105. Dai, J. R., Zhang, Y. P., Jiang, Y. J., Xi, W. P., Yang, G. J. and Liang, Y. S. (2002b). Studies on standardization of methods for screening molluscicides in laboratory. I. Volume of molluscicidal solution to influence the efficacy. Chinese Journal of Schistosomiasis Control 14, 122–124. Dai, J. R., Xi, W. P., Liang, Y. S. and Zhang, Y. P. (2002c). Studies on standardization of methods for screening molluscicides in laboratory. II. Quantity of snails used to influence the molluscicidal efficacy. Chinese Journal of Schistosomiasis Control 14, 263–265. Dai, J. R., Liang, Y. S., Zhang, Y. P., Xu, M., Li, H. J. and Zhu, Y. C. (2003). Studies on standardization of methods for screening molluscicides in laboratory. III. Breeding time of snails to influence the evaluation of the molluscicidal efficacy. Chinese Journal of Schistosomiasis Control 15, 346–348. Dai, J., Li, Y., Wang, W., Xing, Y., Qu, G. and Liang, Y. (2014). Sensitivity of Oncomelania hupensis to niclosamide: a nation-wide survey in China. International Journal of Environmental Research and Public Health 11, 3086–3095. Davis, G. M., Zhang, Y., Guo, Y. H. and Spolsky, C. M. (1995). Population genetics and systematic status of Oncomelania hupensis (Gastropoda: Pomatiopsidae) throughout China. Malacologia 37, 133–156. Engels, D., Chitsulo, L., Montresor, A. and Savioli, L. (2002). The global epidemiological situation of schistosomiasis and new approaches to control and research. Acta Tropica 82, 139–146. Engels, D., Wang, L. Y. and Palmer, K. L. (2005). Control of schistosomiasis in China. Acta Tropica 96, 67–68. Gönnert, R. (1961). Results of laboratory and field trials with the molluscicide Bayer 73. Bulletin of the World Health Organization 25, 483–501. Li, S. Z., Wang, Y. X., Yang, K., Liu, Q., Wang, Q., Zhang, Y., Wu, X. H., Guo, J. G., Bergquist, R. and Zhou, X. N. (2009). Landscape genetics: the correlation of spatial and genetic distances of Oncomelania hupensis, the intermediate host snail of Schistosoma japonicum in mainland China. Geospatial Health 3, 221–231. Li, Y. Z., Xing, Y. T., Li, H. J., Qu, G. L., Wang, W., Wei, J. Y., Liang, Y. S. and Dai, J. R. (2012). Studies on standardization of methods for screening molluscicides in laboratory IV Sensitivity of Oncomelania snails from different months to niclosamide. Chinese Journal of Schistosomiasis Control 24, 35–39. Mao, C. P. (1990). Biology of Schistosome and Control of Schistosomiasis. People’s Medical Publishing House, Beijing, China. McManus, D. P., Li, Y., Gray, D. J. and Ross, A. G. (2009). Conquering ‘snail fever’: schistosomiasis and its control in China. Expert Review of Anti-infective Therapy 7, 473–485. MOH. (2000). Handbook for Schistosomiasis Control in China. Shanghai Scientific and Technology Literature Publishing House, Shanghai, China. Oliveira-Filho, E. C., De-Carvalho, R. R. and Paumgartten, F. J. (1999). The influence of environmental factors on the molluscicidal activity of Euphorbia milii latex. Journal of Environmental Science and Health (Part B) 34, 289–303. Paulini, E. (1958). Bilharziasis control by application of molluscicides; a review of its present status. Bulletin of the World Health Organization 18, 975–988. Steinmann, P., Keiser, J., Bos, R., Tanner, M. and Utzinger, J. (2006). Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infectious Diseases 6, 411–425. Utzinger, J., Zhou, X. N., Chen, M. G. and Bergquist, R. (2005). Conquering schistosomiasis in China: the long march. Acta Tropica 96, 69–96. Wang, L. D., Utzinger, J. and Zhou, X. N. (2008). Schistosomiasis control: experiences and lessons from China. Lancet 372, 1793–1795. Wang, L. D., Guo, J. G., Wu, X. H., Chen, H. G., Wang, T. P., Zhu, S. P., Zhang, Z. H., Steinmann, P., Yang, G. J., Wang, S. P., Wu, Z. D., Wang, L. Y., Hao, Y., Bergquist, R., Utzinger, J. and Zhou, X. N.

Niclosamide resistance in O. hupensis (2009). China’s new strategy to block Schistosoma japonicum transmission: experiences and impact beyond schistosomiasis. Tropical Medicine and International Health 14, 1475–1483. Webbe, G. (1961). Laboratory and field trials of a new molluscicide, Bayer 73, in Tanganyika. Bulletin of the World Health Organization 25, 525–531. World Health Organization (WHO) (1992). The Role of Mollusciciding in Schistosomiasis Control. Division of Control of Tropical Diseases, Geneva, Switzerland. Wilke, T., Davis, G. M., Chen, C. E., Zhou, X. N. and Zeng, X. P. (2000). Oncomelania hupensis (Gastropoda: Rissooidea) in eastern China: molecular phylogeny, population structure, and ecology. Acta Tropica 77, 215–227. Wu, X. Y., Yang, L. Q., Zhang, L. H. and Ge, Q. J. (2006). Progress of research on molluscicides. Chinese Journal of Schistosomiasis Control 18, 474–476. Xianyi, C., Liying, W., Jiming, C., Xiaonong, Z., Jiang, Z., Jiagang, G., Xiaohua, W., Engels, D. and Minggang, C. (2005). Schistosomiasis control in China: the impact of a 10-year World Bank Loan Project (1992–2001). Bulletin of the World Health Organization 83, 43–48. Yang, G. J., Sun, L. P., Hong, Q. B., Zhu, H. R., Yang, K., Gao, Q. and Zhou, X. N. (2012). Optimizing molluscicide treatment strategies in different control stages of schistosomiasis in the People’s Republic of China. Parasites and Vectors 5, 260. Yuan, Y., Xu, X. J., Dong, H. F., Jiang, M. S. and Zhu, H. G. (2005). Transmission control of schistosomiasis japonica: implementation and evaluation of different snail control interventions. Acta Tropica 96, 191–197. Zhang, Y. P., Dai, J. R., Huang, Y. X., Xi, W. P. and Jiang, Y. J. (1999). Sensitivity of Oncomelania hupensis to niclosamide after breeding in laboratory. Journal of Practical Parasitic Diseases 7, 162–164. Zhao, Q. P., Jiang, M. S., Littlewood, D. T. and Nie, P. (2010). Distinct genetic diversity of Oncomelania hupensis, intermediate host of Schistosoma japonicum in mainland China as revealed by ITS sequences. PLOS Neglected Tropical Diseases 4, e611.

9 Zheng, H., Zhang, L. J., Zhu, R., Xu, J., Li, S. Z., Guo, J. G., Xiao, N. and Zhou, X. N. (2012). Schistosomiasis situation in People’s Republic of China in 2011. Chinese Journal of Schistosomiasis Control 24, 621–626. Zhou, P. S., Ma, L. and Huang, J. L. (2002). Research progress on molluscicides and their application. Chinese Journal of Vector Biology and Control 13, 231–233. Zhou, X. N. (2005). Science of Oncomelania Snail. Science Press, Beijing, China. Zhou, X. N., Wang, L. Y., Chen, M. G., Wu, X. H., Jiang, Q. W., Chen, X. Y., Zheng, J. and Utzinger, J. (2005). The public health significance and control of schistosomiasis in China – then and now. Acta Tropica 96, 97–105. Zhou, X. N., Guo, J. G., Wu, X. H., Jiang, Q. W., Zheng, J., Dang, H., Wang, X. H., Xu, J., Zhu, H. Q., Wu, G. L., Li, Y. S., Xu, X. J., Chen, H. G., Wang, T. P., Zhu, Y. C., Qiu, D. C., Dong, X. Q., Zhao, G. M., Zhang, S. J., Zhao, N. Q., Xia, G., Wang, L. Y., Zhang, S. Q., Lin, D. D., Chen, M. G. and Hao, Y. (2007). Epidemiology of schistosomiasis in the People’s Republic of China, 2004. Emerging Infectious Diseases 13, 1470–1476. Zhou, X. N., Bergquist, R., Leonardo, L., Yang, G. J., Yang, K., Sudomo, M. and Olveda, R. (2010). Schistosomiasis japonica control and research needs. Advances in Parasitology 72, 145–178. Zhou, Y. B., Yang, M. X., Zhao, G. M., Wei, J. G. and Jiang, Q. W. (2007). Oncomelania hupensis (Gastropoda: Rissooidea), intermediate host of Schistosoma japonicum in China: genetics and molecular phylogeny based on amplified fragment length polymorphisms. Malacologia 49, 367–382. Zhu, D., Yao, P. and Bao, Z. (1999). Mollusciciding action and toxicity of bromoacetamide. Chinese Journal of Parasitology and Parasitic Diseases 17, 244–246. Zhu, D., Zhou, X. N., Zhang, S. Q., Zhang, G., Liu, H. X., Lu, D. B., Cai, G. Y., Ni, Q. Z., Cao, Z. G. and Wu, W. D. (2006). Study on the molluscicidal effect of META-Li against Oncomelania hupensis. Chinese Journal of Parasitology and Parasitic Diseases 24, 200–203.