INTRODUCTION
Anopheles sinensis Wiedemann is a major vector of human malaria in southeast Asia1–3 and is among the dom- inant mosquito species in Shandong province, China4–6. Shandong province was once an endemic region for ma- laria; in the early 1960s and 1970s7, two large-scale out- breaks occurred with annual infections of six and four mil- lion people, respectively. Due to the large-scale surveys and antimalarial campaigns, the disease burden sharply reduced. At present, the reported malaria incidence has decreased to low levels and imported malaria has become the greatest challenge in Shandong8. Recent malaria out- breaks and re-emergences have been reported only in ar- eas prevalent with An. sinensis6, with peak incidence of 0.019 per 10,000 in the year 2007 in the province9.
Anophelines exhibit a wide range of host preferences including humans, livestock, birds, and reptiles10–12. The risk of malaria transmission and prevalence of malaria are influenced by anopheline mosquitoes’ host selection13-14. Host preferences, especially the degree of anthropophily, affect the efficacy of the disease transmission15. To com- bat malaria, vector control is indispensable in endemic foci16. Hence, vector surveillance is essential to prevent recrudescence of malaria in low-transmission regions17. Identification of blood meals in mosquitoes is an impor- tant step in malaria epidemiological studies18–19. This is a measure of human-vector contact20 and is useful in the estimation of vectorial capacity.
Extensive studies have been done on the feeding be- haviour and blood meal origins of An. sinensis mosquitoes in China (Anhui, Zhejiang, Jiangsu, Henan provinces and
Host preferences and feeding patterns of Anopheles sinensis Wiedemann in three sites of Shandong province, China
Chongxing Zhang
1–2, Guihong Shi
1, Peng Cheng
1, Lijuan Liu
1& Maoqing Gong
11Department of Medical Entomology, Vector Biology Key Laboratory of Medicine and Health Shandong Province, Shandong Institute of Parasitic Diseases, Shandong Academy of Medical Sciences, Jining; 2Collaborative Innovation Center for the Origin and Control of Emerging Infectious Diseases, Taishan Medical University, Taian, Shandong, China
ABSTRACT
Background & objectives: Anopheles sinensis Wiedemann is a major vector of malaria and is among the dominant species in Shandong province of China. Knowledge of the blood-feeding patterns of mosquitoes is crucial for elimination of malaria vectors. However, little information is available on the blood-feeding behaviour of An.
sinensis mosquitoes in Shandong province. This study was carried out to compare the blood-feeding behaviour of An. sinensis in malaria-endemic areas of Shandong province China.
Methods: Adult Anopheles mosquitoes were collected from three malaria-endemic areas (Jimo, Yinan and Shanx- ian), during the peak months of mosquito population (August and September) from 2014 to 2015. Indoor-resting mosquitoes and outdoor-resting blood-fed females were sampled in the morning hours (0600 to 0900 hrs) from 10 randomly selected houses using pyrethrum spray catch method, and sweeping with an insect net. ELISA was used for the identification of blood meal. The blood meal of each mosquito was tested against antisera specific to human, pig, dog, cow, goat, horse (mule) and fowl.
Results: At all indoor study locations of Jimo, Yinan and Shanxian, 59.4, 68.1 and 98.8% blood-engorged female An. sinensis collected from cattle sheds fed almost exclusively on bovines, respectively. For outdoor locations, at Jimo site, 27.27 and 49.55% An. sinensis fed on cattle and pigs; at Yinan, 30.42% fed on cattle and 36.88% fed both on cattle and goats, while no pig antibodies were detected. At Shanxian, percent of An. sinensis that fed on cattle, pigs and cattle-goat was 20.72, 27.62 and 21.78%, respectively.
Interpretation & conclusion: The analysis of An. sinensis blood meals in all the three studied areas from human houses, cattle sheds, pig sheds and mixed dwellings revealed that An. sinensis prefers cattle hosts, and can feed on other available animal hosts if the cattle hosts are absent, and the mosquitoes readily feed on humans when domestic animals (cattle and pigs) are not nearby for feeding. The analysis of blood meal revealed that An. sinensis follow opportunistic feeding in Shandong province, China.
Key words Anopheles sinensis Wiedemann; ELISA; host preference; malaria
Chongqing city), Japan, Korea and Thailand21–30. Though vectorial capacity of An. sinensis have been studied dur- ing 1975–84 and 1996 in Jining district of Shandong31–32, and in 2012 in Huang-Huai Valley of China33; little re- search has been conducted on the transit period of host preferences and blood-feeding behaviour of An. sinensis species from the control phase to the elimination phase in Shandong province, China. Therefore, the present study was conducted to determine the blood feeding behaviour and host preference of the malaria vector, An. sinensis in- habiting the malaria endemic areas of Shandong province for planning appropriate vector control strategy.
MATERIAL & METHODS Study area
The present study was conducted in three malaria- endemic areas in Shandong province of China, character- ized by different levels of past incidence of P. vivax ma- laria. These included a high-risk village, Dachen (34° 49′
10′′ N, 116° 02′ 15′′ E) (Shanxian county, annual average incidence rate >100/100,000), an intermediate risk vil- lage, Gegou (35° 21′ 05′′ N, 118° 29′ 00′′ E) (Yinan coun- ty, annual average incidence rate ~10 to ~100/100,000) and a low-risk village, Xiazhuang (36° 14′ 27′′ N, 120°
27′ 14′′ E) (Jimo county, annual average incidence rate
<10/100,000)34. Altitude of this region is ~70 m. The cli- mate is warm from May to October, and the annual aver- age temperature is 14.3 °C. The range of annual rainfall is 550–1548 mm, and the rainfall concentration is higher from June to September months. The main crops of these villages are wheat, soybean, corn, cotton and potato.
The inhabitants of the villages live in houses that are made of bricks. Additionally, most families in these vil- lages keep domestic animals in their compound such as bovines, dogs, pigs, goats, chickens and others. A typical compound consists of a human residence and a shed for animals. The average distance between domestic animal sheds and human dwellings ranged from 15–20 m. Each of the three villages is surrounded by dense vegetation, which provides potential resting and oviposition sites for mosquitoes. Doors and windows are normally kept open until people go to bed.
The presence of waterbodies distribution and amount of appropriate breeding habitat for An. sinensis larvae was characteristically higher in Dachen village than that in Gegou and Xiazhuang villages. During the study period, there were many ponds and canals that contained endogenous lotus, and a large number of anopheline species larvae were observed in Dachen village.
Ethical approval
The study was initiated after obtaining approved ethical clearance from the Institutional Ethics Committee of the Shandong Institute of Parasitic Diseases (SIPD), Shandong Academy of Medical Sciences, Shandong. The area chief and villagers were sensitized on the study ac- tivities planned, and written consent of authorizing for mosquito collection was obtained.
Mosquito collection
Freshly engorged adult An. sinensis mosquitoes were collected (indoor and outdoor collections at resting places) from three malaria-endemic areas (Jimo, Yinan and Shanxian counties of Shandong province), during the peak months of the mosquito population (August and September) of the years 2014 and 2015. Indoor-resting mosquitoes were sampled during the morning hours (0600 to 0900 hrs) from 10 randomly selected houses by the pyrethrum spray catch method35. At the same time, to study unbiased host feeding patterns of engorged female mosquitoes from vegetation and bushes in the three study areas, outdoor-resting blood-fed females were collected by sweeping with an insect net 36. The collection sites were confined to places 0–250 m away from the houses and the animal sheds around the villages37. The low vegeta- tion that constitutes resting sites was stirred up to disturb mosquitoes, and flying females were caught. Each capture was performed by a single collector and lasted for 30 min.
Fully-fed to half-fed An. sinensis females were used for blood meal identification.
Collected female mosquitoes were individually trans- ferred to 10 ml glass vials, placed on ice using a mouth aspirator, brought to the laboratory, and identified mor- phologically as An. sinensis species38. Mosquitoes were stored at –20°C for blood meal analysis.
Detection of blood meal sources
The stomach contents of freshly engorged An. si- nensis females were dissected out and used as a template for blood-meal analysis. ELISA as previously described by Beier et al39 and modified by Loyola et al40 was used to identify the source of the mosquito’s blood meal. The blood meal of each mosquito was tested against antisera specific to humans, cattle, goats, horses, pigs, dogs, and fowl. All the antisera were obtained from Chinese Cen- ter for Disease Control and Prevention, Government of China.
Statistical analysis
Changes in the host preferences and host feed- ing patterns of An. sinensis on selected host species
and study sites were analyzed by chi-square (χ2) analy- sis, to determine differences using Python (version 3.5) for Windows. The level of significance was set at p < 0.05).
RESULTS Study area
According to the data shared by the village doctors, Dachen village covers approximately 2400 acres of ar- able land, and has a population of 1290 people, 30 calves, and 150 goats in 480 households; Gegou village covers approximately 2500 acres of arable land, with a popula- tion of 1675 people, 60 calves, 70 pigs, and 180 goats in 470 households, and Xiazhuang village covers ap- proximately 1300 acres of arable land, with a population of 950 people, five calves, 45 pigs, and 70 goats in 170 households.
Blood meal analysis result of mosquitoes
The numbers of An. sinensis collected indoors vs outdoors were recorded during the study period (Ta- ble 1). A total of 354 (134 indoors vs 220 outdoors), 417 (154 indoors vs 263 outdoors) and 1487 (734 in- doors vs 753 outdoors) blood-engorged An. sinensis fe- males were collected from Jimo, Yinan and Shanxian, respectively.
The results indicated that the proportion of blood- fed mosquitoes did not differ significantly between those
collected indoors and outdoors in Shanxian county. Sig- nificantly more mosquitoes were collected outdoors than indoors in Yinan and Jimo counties. At all indoor study locations of Jimo, Yinan and Shanxian, 1.4, 23.2 and 83.3% An. sinensis females collected from human houses fed on humans, respectively. This was statistically signifi- cant (χ2 = 177.111, p <0.001). Similarly, 59.4, 68.1 and 98.8% An. sinensis females collected from cattlesheds of Jimo, Yinan and Shanxian fed almost exclusively on bovines, respectively; which was statistically significant (χ2 = 305.772, p <0.001). For outdoor locations, at Jimo site, 27.27 and 49.55% An. sinensis fed on cattle and pigs, respectively. At Yinan, 30.42% An. sinensis females fed on cattle, 36.88% fed on both cattle and goats; while no pig antibodies were detected. At Shanxian, 20.72 and 27.62%
An. sinensis fed on cattle and pigs, respectively, and the percent for feeding cattle-goat was 21.78% (Table 2). For both indoor and outdoor collections, An. sinensis prefer- entially fed on domestic animals rather than humans.
DISCUSSION
The determination of a mosquito blood meal helps us to understand the pathogen’s life cycle and its potential hosts, and to identify suitable control strategies41–43. Host feeding pattern is influenced by numerous factors such as host preference, host availability and host irritability.
Epidemiologically, it is quite important to know the con- ditions of host availability under which species with fixed
Table 1. Ingested-blood meal analysis in An. sinensis collected from human houses and animals' sheds
Areas Location No.
tested Number of blood-meal sources analyzed No-
call*
Human Cattle Goat Pig Dog Fowl Horse/
Donkey/Mule Cross-reaction
Jimo Human houses 58 8 (1.4) 1 (1.7) 0 0 0 0 37 (64) Human/Cow 5 (8.6)
Cattlesheds 69 1 (1.4) 41 (59.4) 0 0 0 0 24 (35) Human/Cow 3 (4.3)
Donkey sheds 7 0 0 0 0 0 0 7 (100) 0
Total 134 9 (6.7) 42 (31.4) 0 0 0 0 68 (51) Human/Cow 8 (5.9)
Yinan Human houses 56 13 (23.2) 1 (1.8) 0 0 0 0 37 (66.1) 0
Cattlesheds 91 1 (1.1) 62 (68.1) 0 0 0 0 24 (26.3) 0
Donkey sheds 7 0 0 0 0 0 0 7 (100) 0
Total 154 14 (9.9) 63 (40.9) 0 0 0 0 68 (44.1) 0
Shanxian Human houses 149 124
(83.3) 9 (6.04) 1
(0.67) 9
(6.04) 2 (1.34) 2
(1.34) 0 Human/Cow 1 (0.67) Human/Pig 1 (0.67) Cattlesheds 166 1 (0.6) 162 (98.8) 0 0 0 0 2 (1.2) Cattle/Goat 1 (0.6) Pig herds 189 0 2 (1.05) 0 184 (97.3) 3(1.58) 0 0 0 Shed with Cattle,
Horse, Mule, Goat 230 0 22
(9.5) 54
(23.5) 0 0 0 148
(64.3) Goat/Cattle 5 (2.1) Total 734 125 (17) 195 (26.6) 55 (7.5) 193 (26.3) 5 (0.6) 2 (0.2) 150 (20.4) 8 (1.1)
*No reaction with antibodies to any of the tested hosts; Figures in parentheses indicate percent positives.
feeding patterns may deviate from their normal hosts44. In a study in Japan, Sasa22 showed that An. hyrca- nus sinensis was strongly attracted to large animals and very few fed on humans. Similarly, study by Otsuru and Ohmori23 showed that cows were the most attractive ani- mal to An. sinensis, but they fed persistently on humans as well. Other studies carried out in Korea have shown that An. sinensis fed exclusively on bovines, though they also fed on dogs, chicken and cats in small numbers, with al- most no feeding on humans, hence human blood meal rate was very low (0.7–1.7%)25, 28–29. In Thailand, An. sinensis was almost entirely zoophilic; almost none of the mosqui- toes were attracted to humans in comparative biting tests involving humans and cows26. In another study carried out by Chow21 in Chongqing, China, indicated that An.
sinensis fed mainly on cows and rarely on humans. Stud- ies in Anhui, Zhejiang and Jiangsu provinces of China have shown that An sinensis feeds both on humans and cattle, but has a strong preference for cattle blood24. Pigs, goats and calves were more attractive to An. sinensis than humans in Yongcheng city of Henan province30.
Indoor collections of this study, at Shanxian site showed that 83.3% of the An. sinensis mosquitoes col- lected from households were positive for human blood, while at Jimo and Yinan, only 1.4 and 23.2% of blood- engorged An. sinensis females mosquitoes showed blood meals positive for human blood, respectively. At Jimo, Yinan and Shanxian sites, 59.4, 68.1 and 98.8% An. sinen- sis females collected from cattle sheds showed blood meal positives for cattle, respectively. Similarly, 27.27, 30.42 and 20.72% female An. sinensis mosquitoes collected outdoors from Jimo, Yinan, Shanxian fed on bovines, respectively. Outdoor collections revealed that 3.98%
(Shanxian), 2.29% (Yinan) and 3.6% (Jimo) mosquitoes were positive for human blood. No pig antibodies were detected at Yinan which might be due to limited number of pig presence. At Shanxian, higher percent of An. sinensis fed on humans than at Jimo and Yinan, the reason could be presence of more waterbodies and appropriate An. si- nensis larvae breeding habitats at Dachen village (Shanx- ian) than those at Gegou village (Yinan) and Xiazhuang
village (Jimo). Thus, host preference of An. sinensis in Shandong province was consistent with other studies in Japan, Korea, Thailand and other provinces in China.
CONCLUSION
The analysis of mosquito blood meals from human households, cattle and pig sheds and mixed dwellings revealed that An. sinensis prefers cattle than pigs, dogs, goats and humans in Shandong province. Additionally, the present study indicates that An. sinensis in the study area is preferentially zoophilic in nature. The results also suggest that the vector can feed on other available hosts, if the cattle host is absent. Though human blood meal rates were low, An. sinensis readily feeds on humans in great numbers when domestic animals (cows and pigs) are not nearby for feeding. This phenomenon could be explained by opportunistic feeding behaviour present in this species.
Though An. sinensis is the most widely distributed species in China, due to its exophilic and exophagic host prefer- ences, the species had been judged not to be the efficiency vector for malaria for decades.
Conflict of interest
The authors declare that they have no competing interests.
ACKNOWLEDGEMENTS
The present study was supported by the National Natural Science Foundation of China (NSFC) (Grant No.
81471985, 81672059) and the Innovation Project of Shan- dong Academy of Medical Sciences, Shandong, China.
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Correspondence to: Dr Chongxing Zhang, Maoqing Gong, Department of Medical Entomology, Vector Biology Key Laboratory of Medicine and Health Shandong Province, Shandong Institute of Parasitic Diseases, Shandong Academy of Medical Sciences, Jining, Shandong–272033, P.R. China.
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