Since the malaria elimination program was launched in China in 2010, the number of local infections has declined from 4,262 in 2010 to none in 2017, indicating remarkable achievements for prevention and treatment (Zhang et al. 2018). Shandong Province is a malaria-endemic area, and vivax malaria is prevalent throughout the province. In 2010, Shandong Province launched the Action Plan to Eliminate Malaria with the implementation of preventative and control measures. Specifically, the “1-3-7” strategy is a simplified set of targets that delineates responsibilities and actions with the following indicators: 1 = case reporting within one day; 3 = case investigation within three days; and 7 = focused investigation and response within seven days. The implementation of the precise management of malaria cases, vector control (indoor residual spraying), and more intensive reactive case detection in each epidemic site caused local cases to decline rapidly. As of 2018 (data from 2018 were not yet published), no local infection cases had been reported in the previous seven consecutive years, indicating that the goal of eliminating malaria had been achieved. However, in recent years, the number of imported malaria cases in Shandong Province has increased substantially, ranking Shandong Province among the highest for malaria cases in China (Feng et al. 2014, Zhang et al. 2017).

Although local infections have been eliminated nationwide, increased foreign aid projects, migrant workers from malaria-endemic areas, and conditions conducive to malaria transmission, especially the complex natural and ecological environment, confer new challenges for malaria elimination. Therefore, to assess the risk of transmission and the epidemiology and trends of malaria, this study analyzed the malaria vector surveillance program in Shandong Province during 2010–2018. The resistance of Anopheles in Huanggang town, where the last local malaria case occurred, was monitored from 2012–2018.

The study was conducted in four cities of Shandong Province, including Hanzhuang town (34°6009N, 117°3568E), Binhu town (35°1426N, 116°9036E), Laohu town (36°0007N, 116°2524E), and Huanggang town (34°6386N, 116°0102E). These towns were chosen because they were once high-risk areas for malaria, are close to the lake, and are densely populated. Hanzhuang town and Binhu town are located beside Weishan Lake, and Laohu town and Huanggang town are located beside Dongping Lake and the Yellow River, respectively. The average annual rainfall for the area is 750 mm, with 60–70% of rainfall occurring from June to August. The districts are rich in water, with rice, wheat, and corn as the predominant crops. Local residents primarily depend on rice and wheat farming, fishing, and livestock rearing for subsistence.

Adult mosquitoes were collected from June to October 2010–2018 in these four towns. Human-baited double-net traps (HDNs) were set up to collect adult mosquitoes at mosquito breeding sites four h after sunset. Eleven breeding sites (beside rice fields, lakes, small pools, ditches, houses, and livestock sheds) in the four study towns were randomly selected, and the same sampling efforts were applied to all towns. Mosquitoes were collected bi-weekly. The same sampling efforts were applied in all towns. Captured mosquitoes were killed with chloroform and identified by morphological characteristics using taxonomic identification keys.

The last local case of malaria occurred in Huanggang town in 2011. Therefore, larvae were collected from potential An. sinensis breeding sites (rice fields, ditches, streams, rivers, etc.) of Huanggang town from 2012–2018. Larval samples were collected by the hand dipper sampling (Silver 2008). The larvae were returned to the laboratory and reared to adulthood. An. sinensis were identified by morphological characterization and used for adult insecticide resistance bioassays. The larvae and adult mosquitoes were fed Tetramin™ fish food and 10% sucrose solutions, respectively.

Female An. sinensis collections were tested for susceptibility to deltamethrin according to WHO guidelines. Twenty to twenty-five female mosquitoes were maintained in recovery tubes for 24 h after exposure to 0.05% deltamethrin insecticide-impregnated papers (Penang, Malaysia) for 1 h. Then, the numbers of surviving and dead mosquitoes were counted. Silicone oil-treated papers were used as a control (Penang, Malaysia) using the same protocol. Five replicates were performed for each mosquito sample.

The density of anopheline mosquitoes was calculated as the number of adult anopheline mosquitoes per human collector per hour. The mortality rate was calculated as the number of dead mosquitoes per number of tested mosquitoes. When the control mortality was ≥20%, the tests were invalidated. If the control mortality was <20%, the mortality was corrected by Abbott's formula. According to the resistance interpretation of the WHO in 2013, a mortality rate of 98–100% was classified as susceptible (S), 90–97% was classified as suspected resistant (SR), and less than 90% was classified as resistant (R). Analysis of variance (ANOVA) was used to test the association of different months and years with vector density. Data were analyzed using SPSS version 19.0 software, and P < 0.05 was considered statistically significant.

A total of 7,309 Anopheles was collected from 2010–2018 (Table 1). All of the captured Anopheles mosquitoes were An. sinensis. An. sinensis density was calculated as the number of adult mosquitoes per person per hour (Figure 1). The density of An. sinensis was 0–13.8 mosquitoes per person per hour, and the highest density occurred in August, 2016. There was no significant difference in adult An. sinensis densities in different years (Welch: F = 0.341, P = 0.936). However, there was a significant difference in An. sinensis density in different months from 2010–2018 (Welch: F = 39.734, P < 0.001). The mosquito densities in July and August were significantly higher than those in other months (all P < 0.05), but no significant difference was found between July and August (P = 0.679). An. sinensis was mostly prevalent in July and August of 2010–2018.

The resistance of An. sinensis from Huanggang town to 0.05% deltamethrin during 2012 to 2018 is presented in Table 2. Adult bioassays of An. sinensis consistently showed resistance to deltamethrin, and the mean difference in mortality rate was 46.5% (95% CI 42.1–50.9%) from 2012–2018. The lowest mortality was observed in 2012 (mortality: 39.4±9.0%), while the highest mortality was in 2014 (mortality: 50.9±8.4%). There was no significant difference in the adult bioassay results in different years (F = 1.478, P = 0.222).

In this study, all captured Anopheles mosquitoes were An. sinensis, which is the most important vector for the spread of Plasmodium vivax malaria in Shandong Province. In contrast to our research, Dai et al. (2011) observed a low quantity of Anopheles lesteri in Shandong Province in 2007–2009, which is also a vector of P. vivax and Plasmodium falciparum. However, An. lesteri was not captured in our research and has not been captured in Jiangsu Province since 2001. This indicates that An. sinensis is the predominant malaria vector in Shandong Province, whereas An. lesteri and An. anthropophagus populations have decreased and likely play a negligible role in malaria transmission. Similar results were reported for An. lesteri and An. anthropophagus, which have disappeared in the neighboring Jiangsu Province (Li et al. 2018) and Anhui Province (Liu Feng et al. 2017). Moreover, national surveillance (Feng et al. 2014) showed that An. sinensis accounted for more than 83% of the Anopheles populations at the monitoring sites, and other Anopheles mosquitoes accounted for a relatively low proportion. This phenomenon may be related to monitoring methods and species distribution changes due to environmental changes caused by China's economic development (Feng et al. 2014).

Similar results from previous studies showed that An. sinensis appeared in May and disappeared in October, with a peak density occurring between July and August (Li et al. 2018). Thus, in order to prevent malaria transmission, more attention should be paid to preventing and reducing mosquito bites in July and August due to high mosquito densities. The density of An. sinensis was 0–13.9 mosquitoes per person per hour in Shandong Province. The density of An. sinensis reached 14.2 mosquitoes per person per hour in Anhui Province (Feng et al. 2014) and 25.03 mosquitoes per person per hour in Jiangsu Province (Li et al. 2018). Studies have shown that the peak biting time of An. sinensis occurs from 19:00–20:00 (Burkot et al. 2018); consequently, personal protection should be enhanced during this time. The density of An. sinensis at the pre-elimination and elimination stages did not obviously change. However, An. sinensis acted as the single malaria transmission vector and monitoring should still be strengthened.

The resistance level of An. sinensis to deltamethrin was consistently evaluated as R since the last local case of malaria that occurred in Shandong Province 2012 – 2018. Regarding deltamethrin resistance, Anopheles were initially evaluated as having suspected resistance (Dai et al. 2015) (mortality: 84.2%, according to the WHO criteria in 1998; mortality: 80–97% prior to 2004) and then were classified as resistant (Dai et al. 2015) (mortality: 73.6%) after 2005 and have remained resistant ever since. However, the mortality rate declined to 58.3% in 2006 (Dai et al. 2015). In addition, the reduction in mortality doubled over ten years, ranging from 81.4% in 2003 (Dai et al. 2015) to 39.4% in 2012. Coincidentally, there was a malaria peak in Shandong Province during 2006–2009 (Kong et al. 2017). During this period, anti-malarial measures were intensified, including indoor residual spraying, environmental improvements, and the distribution of insecticide-treated nets. The wide use of pesticides may increase the resistance of mosquitoes. An. sinensis also showed high resistance to pyrethroid insecticides in Hainan Province, Yunnan Province, Anhui Province (Chang et al. 2014), and Guangxi Province. This investigation revealed that deltamethrin resistance could be prevalent and accentuated by the exposure of mosquito populations to pesticides used in vector control and agriculture. In addition, field An. sinensis populations from Shandong also displayed high resistance to DDT, cyfluthrin, and malathion (Dai et al. 2015). The resistance status of An. sinensis is still serious.

By 2020, malaria will be considered to be eliminated in China (Lei and Wang 2012). Although locally transmitted malaria cases have been eliminated in Shandong since 2012, imported cases have exhibited an increasing trend from 93 in 2012 to 212 in 2015 (Kong et al. 2017). Most of these patients returned from Equatorial Guinea and Angola. Considering the dense local human populations, increasing mobility of human populations, vector competence of field An. sinensis, mosquito population density, structure variations caused by environmental ecological changes, and the risk posed by insecticide resistance, continuous monitoring of insecticide resistance in malaria vectors and imported cases will help to move the epidemic surveillance gate forward.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (81702034(HML), 81672059(MQG), 81871685(MQG)), the Key Research and Development Program of Shandong Province (2019GSF111006 (PC)).