Ecological niches and blood sources of sand fly in an endemic focus of visceral leishmaniasis in Jiuzhaigou, Sichuan, China
© Chen et al. 2016
Received: 8 December 2015
Accepted: 5 April 2016
Published: 13 April 2016
Sand fly Phlebotomus chinensis is a principle vector for the visceral leishmaniasis (VL) in China with a wide geographic distribution. Jiuzhaigou, Sichuan is a mountain type endemic area of VL in China. Long term effective control efforts in the region have successfully reduced VL transmission. To assess the current status of the sand flies and their ecological aspects in the region, a survey was conducted in the summer of 2014 and 2015.
Sand fly specimens were collected by light traps in a village and blood sources were identified by PCR and sequencing of the mitochondrial cytochrome b gene.
In a rock cave, 65.2 %–79.8 % of collected sand flies were male. On a rabbit farm, 92.9 %–98.8 % of specimens were female. In pig pens, 61.1 % of specimens were female. Some females had visible blood residues. The feeding rate was 49.4 % from the pig pens, 12.3 % from the cave, and only 1.7 % from the rabbit farm. Pig, rabbit, chicken, dog, and human blood were detected in the fed specimens. Swine blood, present in all tested samples, was a preferred blood source, while chicken and dog blood were present in a third of the samples.
In Jiuzhaigou County, Sichuan Province of China, the considerable sandfly density and the peridomestic feeding behavior all increases the risk of VL transmission, and insecticide spraying in animal sheds could be exploited to reduce sand fly populations in human surroundings.
KeywordsSand fly Phlebotomus chinensis Ecological niche Blood sources China
Please see additional file 1 for translation of the abstract into the six official working languages of the United Nations.
Visceral leishmaniasis (VL) is a disease caused by trypanosomatid protozoa in the genus Leishmania and is transmitted by vector species of phlebotomine sand flies. At present, VL is largely endemic in western China; focal and sporadic cases occur in Xinjiang, Inner Mongolia, Gansu, Sichuan, Shaanxi, and Shanxi [1, 2]. Jiuzhaigou is one of the VL endemic foci in Sichuan Province, and sand flies in the region have been investigated since the 1980s [3–6]. Five species of sand flies exist in the area: Phlebotomus chinensis, Ph. sichuanensis, Sergentomyia quamirostris, S. suni, and S. koloshanensis. Phlebotomus chinensis is the most abundant species, accounting for 96 % of the sand fly population [5, 6]. Annually, sand flies emerge in May, peak between late July to early August, and then decline in September and disappear by late October. They are largely exophilic and are commonly found in rock and dirt caves [5, 6]. Epidemiologically, VL in Sichuan is zoonotic, maintained in cycles between animals and sand flies . Phlebotomus chinensis is the principle vector and domestic dogs are the primary reservoir host. Natural infection of Leishmania was detected in wild caught sand fly females with 1.98 % prevalence . The prevalence of Leishmania infection in dogs is high in the region. In two surveys conducted in 2010, the infection rate of Leishmania in dogs at Jiuzhaigou was 59.4 %  and 24.1 % .
Integrated implementations of control efforts in the past decades have greatly reduced the prevalence and incidence of VL in China . In Jiuzhaigou, VL has declined from 60–70 cases a year in the 1970s to less than 10 cases a year in the year 2010–2014. The successful reduction of VL in the region was largely attributed to the control and treatment of infected dogs. Both veterinary care and insecticide-impregnated collars effectively intervened the VL transmission. However, the risk of VL remains due to the existence of wild animal reservoirs and sand flies. Jiuzhaigou is a famous scenic attraction for tourists with approximately 4.5 million visitors in 2014 according to a press release by Jiuzhaigou Tourism Bureau. The non-immune tourists are vulnerable, and risks of contracting VL are persistently present. Surveillance and control of sand flies have become an urgent necessity in the local VL control program. Understanding the current status of the bionomics of sand flies will facilitate development of effective control measures. In this paper, we report the habitat types and blood sources of sand flies in the region.
This study was carried out in strict accordance with the NSFC, NIH and NMSU ethical guidelines for biomedical research involving living animals and human subjects.
Sand fly collection and species identification
Blood source identification
PCR primers for blood source identification
Amplicon length (bp)
GGT TGT CCT CCA ATT CAT GTT A
Human (Homo sapien)
HF + UR
GGC TTA CTT CTC TTC ATT CTC TCC T
Pig (Sus domesticus)
PF + UR
CCT CGC AGC CGT ACA TCT C
Cow (Bos taurus)
CF + UR
CAT CGG CAC AAA TTT AGT CG
Dog (Canis lupus)
DF + UR
GGA ATT GTA CTA TTA TTC GCA ACC AT
Chicken (Gallus gallus)
CAT ACT CCC TCA CTC CCC CA
CCC CTC AGG CTC ACT CTA CT
Rabbit (Oryctolagus cuniculus)
CGA TAC CTC CAC GCT AAC GG
TTG GGT TGT TGG AGC CAG TT
The sex composition was compared between different collections by Chi-Square test, which was conducted by SigmaStat 3.5 (Systat Software Inc.). The feeding rates among different collections were compared by a Chi-Square (r × c) contingency table, which was implemented at http://www.physics.csbsju.edu/stats/contingency_NROW_NCOLUMN_form.html.
Sand fly collections from different ecological niches
Sand fly collections in the study
No. of males (%)
No. of females (%)
Feeding rates in the 2015 collection
No. of fed females (%)
Total No. of females
Blood sources detected in the fed sand flies
(No. of positive samples/total No. of samples)
Sand flies can adapt to various ecological niches and have quite a broad range of hosts as blood sources [12, 13, 15, 16]. The development of an effective measure of sand fly control would largely rely on the understanding of habitats and host preference in a region. In the study, sand flies were sampled from one large rock cave, two rabbit sheds, and three pig pens; which represented three types of habitats in the region. As shown in Table 2, more sand flies were caught in the cave than in the village (rabbit sheds and pig pens). Evidently, the cave was a good breeding habitat close to the village. Males were predominant in the cave collections, suggesting that males stay primarily at that habitat. Females need to hunt for blood in a larger radius. Consistent with this, 12.3 % of females caught in the cave had visible blood residue. Pig, rabbit, chicken, and dog blood were detected in the cave collection. Apparently females fly into the village to obtain blood and return to the cave to lay eggs. It was particularly interesting that rabbit blood was positive in the flies from the cave. There was only one rabbit farm located approximately 500 m away from the cave. Most likely, the sand flies that took rabbit blood would fly to the cave for oviposition. Alternatively, sand flies could have taken blood from wild hares that were near the cave. Overall, the evidences strongly suggest that the rock cave is an optimal sand fly breeding site near the village.
The rabbit sheds were attractive for females most likely due to the high amount of CO2 produced by the large number of rabbits, as CO2 has been shown to be an effective attractant for sand flies [17–19]. Interestingly, the feeding rate in the collection was much lower than from the rock cave and pig pens. We do not have an explanation for this phenomenon. The rabbit and hare have been shown to be a blood source for Phlembotomine sand flies [20–25]. In a focus of leishmaniasis in the southwestern Madrid region, Spain , rabbits may play a role in the transmission of Leishmania infantum to Ph. perniciosus . Potentially, rabbit farming may pose a risk in leishmaniasis endemic areas.
In the collection from pig pens, greater than half specimens were females, and half of the females were engorged. As expected, swine blood was found in all 9 samples from pig pens. In addition, swine blood was found in all 13 samples from the cave and rabbit sheds. It appears that sand flies preferred to take blood from pigs. Pigs have been reported to be a blood source for sand flies . In addition, the soil in the pig pens enriched with organic compounds released from swine excretions may provide supports for larval development [27, 28]. A Leishmania infected pig has been documented. The Leishmania amastigotes were detected in the cutaneous lesion , which left a possibility that pigs might be able to sustain cutaneous infections. In a study conducted in a region of Brazil where American visceral leishmaniasis was endemic, the prevalence of antibodies against L. infantum were about 40 % in the pigs tested. However, when sows were experimentally inoculated with infective L. infantum promastigotes, anti- L. infantum antibody was induced, but no full infection was established. . The data suggest that pigs are able to develop effective immunity to eliminate L. infantum infection. The immunity of pigs against L. infantum infection greatly reduces the possibility of serving as a reservoir host for L. infantum. Recently, multiple lines of evidence suggest that there are heterogeneous Leishmania strains in China. These strains are distinct from but phylogenetically related to L. donovani/L. infantum complex [7, 31–34]. Therefore, further study is needed to investigate whether or not pigs can serve as a reservoir host of the Leishmania strains in China.
The presence of various ecological niches and the availability of ample blood sources from domestic animals contributed to the maintenance of a large population of sand flies. Habitats such as rock caves in the vicinity and peridomestic pigsties should be included in sand fly control. Sand flies in the region were susceptible to insecticides. In 1994, the rock cave was treated with alpha-cypermethrin. The treatment eliminated the sand flies in the cave instantly, and no sand flies were found for four consecutive years . Therefore, spraying residual insecticides inside rock caves and pig pens may be an affordable and sustainable method for reducing the sand fly populations in and around human living quarters.
We are especially grateful to Xiangyu Li providing field assistance in 2014. This work was supported by the YM’s grant 81371848 from the National Natural Sciences Foundation of China; JX’s grants SC1GM109367 from the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health and the DMS-1222592 from National Science Foundation. This work was a part of JX’s sabbatical research in the fall of 2014. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health and National Science Foundation.
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- Wang JY, Cui G, Chen HT, Zhou XN, Gao CH, Yang YT. Current epidemiological profile and features of visceral leishmaniasis in people's republic of China. Parasit Vectors. 2012;5:31. doi:10.1186/1756-3305-5-31.View ArticlePubMedPubMed CentralGoogle Scholar
- Lun ZR, Wu MS, Chen YF, Wang JY, Zhou XN, Liao LF, et al. Visceral Leishmaniasis in China: an Endemic Disease under Control. Clin Microbiol Rev. 2015;28(4):987–1004. doi:10.1128/CMR.00080-14.View ArticlePubMedGoogle Scholar
- Guan L, Gu D, Wang J. Biology and control strategy of phlebotomine sandfly. Int J Med Parasit Dis. 2007;34(6):283–90.Google Scholar
- Xiong G, Jin C. Studies on the longitudinal distibution of sandfly Phlebotomus chinensis and its relation to Kala Azar in southern Gansu and northern Sichuan. Endemic Diseases Bulletin. 1989;4(4):19–21.Google Scholar
- Zhang Y. Investigation of Phlebotomus chinensis in Jiuzhaigou County, Sichuan Province. Int J Med Parasit Dis. 2007;34(5):240–1.Google Scholar
- Jin C, Xiong G, Hong Y, Su Z, Li G, Gao B. Studies on the bionomics of Phlebotomus chinensis in mountain caves and its relation to control in northern Sichuan. Chin J Parasitol Parasitic Dis. 1995;13(4):273–6.Google Scholar
- Wei F, Shang L, Jin H, Lian H, Liu W, Li Z, et al. Molecular detection and genetic diversity of Leishmania donovani in naturally infected Phlebotomus chinensi from southwestern China. Vector Borne Zoonotic Dis. 2011;11(7):849–52. doi:10.1089/vbz.2010.0148.View ArticlePubMedGoogle Scholar
- Wang JY, Ha Y, Gao CH, Wang Y, Yang YT, Chen HT. The prevalence of canine Leishmania infantum infection in western China detected by PCR and serological tests. Parasit Vectors. 2011;4:69. doi:10.1186/1756-3305-4-69.View ArticlePubMedPubMed CentralGoogle Scholar
- Shang LM, Peng WP, Jin HT, Xu D, Zhong NN, Wang WL, et al. The prevalence of canine Leishmania infantum infection in Sichuan Province, southwestern China detected by real time PCR. Parasit Vectors. 2011;4(1):173. doi:10.1186/1756-3305-4-173.View ArticlePubMedPubMed CentralGoogle Scholar
- Lu B, Wu H. Classification and identification of important medical insects of China. Henan: Henan Science and Technology Publishing House; 2003.Google Scholar
- Zhang L, Ma Y. Identification of Phlebotomus chinensis (Diptera: Psychodidae) inferred by morphological characters and molecular markers. Entomotaxonomia 2012;34(1):71–80.
- Abbasi I, Cunio R, Warburg A. Identification of blood meals imbibed by phlebotomine sand flies using cytochrome b PCR and reverse line blotting. Vector Borne Zoonotic Dis. 2009;9(1):79–86. doi:10.1089/vbz.2008.0064.View ArticlePubMedGoogle Scholar
- Garlapati RB, Abbasi I, Warburg A, Poche D, Poche R. Identification of bloodmeals in wild caught blood fed Phlebotomus argentipes (Diptera: Psychodidae) using cytochrome b PCR and reverse line blotting in Bihar, India. J Med Entomol. 2012;49(3):515–21.View ArticlePubMedGoogle Scholar
- Maia C, Parreira R, Cristovao JM, Freitas FB, Afonso MO, Campino L. Molecular detection of Leishmania DNA and identification of blood meals in wild caught phlebotomine sand flies (Diptera: Psychodidae) from southern Portugal. Parasit Vectors. 2015;8:173. doi:10.1186/s13071-015-0787-4.View ArticlePubMedPubMed CentralGoogle Scholar
- Ready PD. Biology of phlebotomine sand flies as vectors of disease agents. Annu Rev Entomol. 2013;58:227–50. doi:10.1146/annurev-ento-120811-153557.View ArticlePubMedGoogle Scholar
- Warburg A, Faiman R. Research priorities for the control of phlebotomine sand flies. J Vector Ecol. 2011;36 Suppl 1:S10–6. doi:10.1111/j.1948-7134.2011.00107.x.View ArticlePubMedGoogle Scholar
- Hoel DF, Zollner GE, El-Hossary SS, Fawaz EY, Watany N, Hanafi HA, et al. Comparison of three carbon dioxide sources on phlebotomine sand fly capture in Egypt. J Med Entomol. 2011;48(5):1057–61.View ArticlePubMedGoogle Scholar
- Beavers GM, Hanafi HA, Dykstra EA. Evaluation of 1-octen-3-ol and carbon dioxide as attractants for Phlebotomus papatasi (Diptera: Psychodidae) in southern Egypt. J Am Mosq Control Assoc. 2004;20(2):130–3.PubMedGoogle Scholar
- Kirstein OD, Faiman R, Gebreselassie A, Hailu A, Gebre-Michael T, Warburg A. Attraction of Ethiopian phlebotomine sand flies (Diptera: Psychodidae) to light and sugar-yeast mixtures (CO(2)). Parasit Vectors. 2013;6(1):341. doi:10.1186/1756-3305-6-341.View ArticlePubMedPubMed CentralGoogle Scholar
- Cotteaux-Lautard C, Leparc-Goffart I, Berenger JM, Plumet S, Pages F. Phenology and host preferences Phlebotomus perniciosus (Diptera: Phlebotominae) in a focus of Toscana virus (TOSV) in South of France. Acta Trop. 2016;153:64–9. doi:10.1016/j.actatropica.2015.09.020.View ArticlePubMedGoogle Scholar
- Gonzalez E, Gallego M, Molina R, Abras A, Alcover MM, Ballart C, et al. Identification of blood meals in field captured sand flies by a PCR-RFLP approach based on cytochrome b gene. Acta Trop. 2015;152:96–102. doi:10.1016/j.actatropica.2015.08.020.View ArticlePubMedGoogle Scholar
- Jimenez M, Gonzalez E, Iriso A, Marco E, Alegret A, Fuster F, et al. Detection of Leishmania infantum and identification of blood meals in Phlebotomus perniciosus from a focus of human leishmaniasis in Madrid, Spain. Parasitol Res. 2013;112(7):2453–9. doi:10.1007/s00436-013-3406-3.View ArticlePubMedGoogle Scholar
- Jimenez M, Gonzalez E, Martin-Martin I, Hernandez S, Molina R. Could wild rabbits (Oryctolagus cuniculus) be reservoirs for Leishmania infantum in the focus of Madrid, Spain? Vet Parasitol. 2014;202(3-4):296–300. doi:10.1016/j.vetpar.2014.03.027.View ArticlePubMedGoogle Scholar
- Martin-Martin I, Molina R, Rohousova I, Drahota J, Volf P, Jimenez M. High levels of anti-Phlebotomus perniciosus saliva antibodies in different vertebrate hosts from the re-emerging leishmaniosis focus in Madrid, Spain. Vet Parasitol. 2014;202(3-4):207–16. doi:10.1016/j.vetpar.2014.02.045.View ArticlePubMedGoogle Scholar
- Johnson RN, Ngumbi PM, Mwanyumba JP, Roberts CR. Host feeding preference of Phlebotomus guggisbergi, a vector of Leishmania tropica in Kenya. Med Vet Entomol. 1993;7(3):216–8.View ArticlePubMedGoogle Scholar
- Morrison AC, Ferro C, Tesh RB. Host preferences of the sand fly Lutzomyia longipalpis at an endemic focus of American visceral leishmaniasis in Colombia. Am J Trop Med Hyg. 1993;49(1):68–75.PubMedGoogle Scholar
- Ferro C, Pardo R, Torres M, Morrison AC. Larval microhabitats of Lutzomyia longipalpis (Diptera: Psychodidae) in an endemic focus of visceral leishmaniasis in Colombia. J Med Entomol. 1997;34(6):719–28.View ArticlePubMedGoogle Scholar
- Moreira Jr ED, de Souza VM, Sreenivasan M, Lopes NL, Barreto RB, de Carvalho LP. Peridomestic risk factors for canine leishmaniasis in urban dwellings: new findings from a prospective study in Brazil. Am J Trop Med Hyg. 2003;69(4):393–7.PubMedGoogle Scholar
- Brazil RP, Desterro MD, Nascimento SB, Macau RP. [Natural infection of a pig (Sus scrofa) by Leishmania in a recent focus of cutaneous leishmaniasis on the Island of Sao Luis, Maranhao]. Mem Inst Oswaldo Cruz. 1987;82(1):145.View ArticlePubMedGoogle Scholar
- Moraes-Silva E, Antunes FR, Rodrigues MS, da Silva JF, Dias-Lima AG, Lemos-de-Sousa V, et al. Domestic swine in a visceral leishmaniasis endemic area produce antibodies against multiple Leishmania infantum antigens but apparently resist to L. infantum infection. Acta Trop. 2006;98(2):176–82. doi:10.1016/j.actatropica.2006.04.002.View ArticlePubMedGoogle Scholar
- Sun K, Guan W, Zhang JG, Wang YJ, Tian Y, Liao L, et al. Prevalence of canine leishmaniasis in Beichuan County, Sichuan, China and phylogenetic evidence for an undescribed Leishmania sp. in China based on 7SL RNA. Parasit Vectors. 2012;5:75. doi:10.1186/1756-3305-5-75.View ArticlePubMedPubMed CentralGoogle Scholar
- Yang BB, Chen DL, Chen JP, Liao L, Hu XS, Xu JN. Analysis of kinetoplast cytochrome b gene of 16 Leishmania isolates from different foci of China: different species of Leishmania in China and their phylogenetic inference. Parasit Vectors. 2013;6:32. doi:10.1186/1756-3305-6-32.View ArticlePubMedPubMed CentralGoogle Scholar
- Yang BB, Guo XG, Hu XS, Zhang JG, Liao L, Chen DL, et al. Species discrimination and phylogenetic inference of 17 Chinese Leishmania isolates based on internal transcribed spacer 1 (ITS1) sequences. Parasitol Res. 2010;107(5):1049–65. doi:10.1007/s00436-010-1969-9.View ArticlePubMedGoogle Scholar
- Zhang CY, Lu XJ, Du XQ, Jian J, Shu L, Ma Y. Phylogenetic and evolutionary analysis of Chinese Leishmania isolates based on multilocus sequence typing. PLoS One. 2013;8(4):e63124. doi:10.1371/journal.pone.0063124.View ArticlePubMedPubMed CentralGoogle Scholar