Phlebotomine sand flies are established vectors of leishmaniasis in humans. In Thailand, Leishmania martiniquensis and “Leishmania siamensis” have been described as causative agents of leishmaniasis. In this study, a survey of sand flies in the Leishmania infected area of Hang Dong district, Chiang Mai, Thailand was performed using CDC light traps for eight consecutive months, from January to August 2016. A total of 661 sand flies were collected, and of 280 female sand flies, four species of the genus Sergentomyia including Sergentomyia gemmea, S. barraudi, S. indica, and S. hivernus and one species of the genus Phlebotomus, Phlebotomus stantoni, were identified. S. gemmea and S. hivernus were found in Chiang Mai for the first time. The density of captured female sand flies was high in warm and humid periods from June to August, with temperatures of around 26°C and relative humidity about 74%. In addition, S. gemmea was the most predominant species in the area. Further studies as to whether or not these sand fly species could be a vector of Leishmaniasis in Thailand are required

Apiwathnasorn, C.

Bates, P. A.

Jariyapan, N.

Mano, C.

Samung, Y.

Siriyasatien, P.

Somboon, P.

Sor-Suwan, S.

Sriwichai, P.

Lancaster E-Prints

1	  	  Species composition and population dynamics of phlebotomine sand flies in a Leishmania infected area of Chiang Mai, Thailand  Sor-suwan, S.1, Jariyapan, N.1*, Mano, C.1, Apiwathnasorn, C.2, Sriwichai, P.2, Samung, Y.2, Siriyasatien, P.3, Bates, P.A.4 and Somboon, P.1* 1Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand 2Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand 3Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 4Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, United Kingdom *Corresponding author email: njariyapan@gmail.com and pradya.somboon@cmu.ac.th   2	  	  Abstract. Phlebotomine sand flies are established vectors of leishmaniasis in humans. In Thailand, Leishmania martiniquensis and “Leishmania siamensis” have been described as causative agents of leishmaniasis. In this study, a survey of sand flies in the Leishmania infected area of Hang Dong district, Chiang Mai, Thailand was performed using CDC light traps for eight consecutive months, from January to August 2016. A total of 661 sand flies were collected, and of 280 female sand flies, four species of the genus Sergentomyia including Sergentomyia gemmea, S. barraudi, S. indica, and S. hivernus and one species of the genus Phlebotomus, Phlebotomus stantoni, were identified. S. gemmea and S. hivernus were found in Chiang Mai for the first time. The density of captured female sand flies was high in warm and humid periods from June to August, with temperatures of around 26°C and relative humidity about 74%. In addition, S. gemmea was the most predominant species in the area. Further studies as to whether or not these sand fly species could be a vector of Leishmaniasis in Thailand are required.  Keywords. Sand flies; Leishmania; Phlebotomine; Thailand 3	  	  INTRODUCTION Leishmaniases are a group of diseases caused by Leishmania parasites occurring in three main forms in humans: visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis (MCL). Related health problems exist in both tropical and subtropical areas, with an estimated 1.3 million new cases and 20,000 to 30,000 deaths occurring annually (WHO, 2016). In Thailand Leishmania martiniquensis and “Leishmania siamensis” have been described as causative agents of leishmaniasis and a leishmaniasis case has been reported in the Hang Dong district, Chiang Mai, Thailand (Chiewchanvit et al., 2015). Sand flies have been suspected for some time of being vectors of leishmaniasis in the country (Kanjanopas et al., 2013; Chusri et al., 2014). Survey studies of sand fly species distribution have been conducted in the central, western, northern, and northeastern regions of Thailand, where four genera, i.e., Sergentomyia, Phlebotomus, Chinius, and Idiophlebotomus have been identified. Of the four genera, Sergentomyia has been the most predominant genus found in all of the study areas. To date, at least 29 sand fly species in several provinces of Thailand have been reported, i.e., Chinius barbazani, Idiophlebotomus teshi, Phlebotomus argentipes, P. asperulus, P. barguesae, P. betisi, P. hoepplii, P. major, P. mascomai, P. phillipinensis, P. pholetor, P. stantoni, Sergentomyia anodontis, S. bailyi, S. barraudi, S. brevicaulis, S. dentata, S. gemmea, S. hodgsoni, S. indica, S. iyengari, S. perturbans, S. phasukae, S. punjabensis, S. quatei, S. sylvatica, (reviewed by Polseela et al., 2016b), S. phadongensis (Polseela et al., 2016a) S. hivernus, and S. khawi (Phumee et al., 2016). Eight of these species have been reported in Chiang Mai, i.e., P. argentipes, P. stantoni, S. anodontis, S. bailyi, S. barraudi, S. indica, S. iyengari, and S. sylvatica (Polseela, 2012). However, little is known about the distribution of sand fly species in Hang Dong, Chiang Mai, Thailand, where leishmaniasis 4	  	  has been reported. Thus, the objective of this study was to identify the distribution of sand fly species and their population dynamics in the infected area.  MATERIALS AND METHODS Study area The Hang Dong District, Chiang Mai, Thailand (18°41′15ʺN et 98°55′28ʺE) was selected as the study area because a leishmaniasis patient had been reported there previously (Chiewchanvit et al., 2015) (Fig. 1).   Sand fly collections Sand flies were collected monthly from January to August 2016 using five CDC light-traps from 6 pm to 6 am (overnight) for two consecutive nights. Annual temperature and relative humidity in the area ranged from 17 to 36°C and 32 to 74.4%, respectively. Collected sand flies were transferred to the laboratory at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, for species identification and investigation for entomopathogenic nematodes.  Sand fly identification All of the sand flies were separated by sex and the numbers of males and females were recorded. Then, they were preserved in 75% ethanol until identification. For sand fly identification, the samples were dissected in Phosphate Buffer Saline (PBS; 10 mM sodium phosphate, 145 mM sodium chloride, pH 7.2) under a dissecting binocular microscope. However, only the female heads and segments 8-11 were cut and mounted in Hoyer’s medium. Sand fly identification was performed using the following keys and articles (Theodor, 1938; Quate, 1962; Lewis, 1978, 1987). 5	  	   Light microscopy The samples were observed under a light microscope and photographed using an OLYMPUS microscopy camera using DP2-SAL Firmware Ver.3.3.1.198 (Tokyo, Japan).  RESULTS In this study, a total of 661 phlebotomine sand flies were found belonging to two genera, Sergentomyia and Phlebotomus with a female:male ratio of 1:1.36. Five species were identified from 280 females, i.e., S. gemmea (35.36%), P. stantoni (28.21%), S. barraudi (18.57%), S. indica (17.14%), and S. hivernus (0.71%) (Table 1). S. gemmea and S. hivernus were found in Chiang Mai for the first time. At this site, S. gemmea was found to be the most abundant species whereas the least common species was S. hivernus. Figure 2 shows the morphological characteristics of the cibarium of the females from the species: P. stantoni, S. gemmea S. barraudi, S. indica, and S. hivernus which were used for species identification in this study. The cibarium of S. gemmea contained 10-12 hind teeth with two or three rows of fore teeth (Fig. 2a) whereas the cibarium of S. hivernus contained 13-14 hine teeth and one row of fore teeth (Fig. 2b). The cibarium of S. indica had 40 teeth in a convex plate with a brown pigment patch (Fig. 2c). The number of teeth of S. barraudi appeared to vary from about 45 to 70 teeth in a straight plate and the pigment patch was bifid (Fig. 2d). P. stantoni had a cibarium with about 15 pointed denticles of various lengths, irregularly arranged, the two or three median ones longer and stouter than the others (Figure 2e). Species identification was also performed using the spermathecae of the females of P. stantoni, S. gemmea S. barraudi, S. indica, and S. hivernus (Fig. 3). Spermathecae of S. gemmea had a narrow shape with knob in a deep narrow pit (Fig. 3a) but spermatheca of S. hivernus had a long, smooth and tubular shape (Fig. 3b). S. indica had a round finely 6	  	  speculate capsule spermatheca with minute projections at its tip (Fig. 3c). Spermathecae of S. barraudi were ovoid and smooth without setae (Fig. 3d). Spermathecae of P. stantoni were fusiform, with 15 of 16 rings, the neck was thick and short, the head more or less oblong (Fig. 3e). Figure 4 shows variation of the sand fly density in each month in the study area. The density of captured female sand flies was high in warm and humid periods from June to August. The greatest number of collected specimens was in June with the temperature around 26°C and relative humidity around 74%. However, P. stantoni, S. gemmea, and S. barraudi were found throughout this study.  DISCUSSION Results in the current study provided information on the distribution of sand fly species in Hang Dong District, Chiang Mai, Thailand where the leishmaniasis case was reported (Chiewchanvit et al., 2015). Eight sand fly species have been reported in Chiang Mai but the suspected vector S. gemmea, and S. hivernus, have not been found before (reviewed by Polseela, 2012). In this study, the identification of sand flies species was performed by using morphological characteristics of abdominal hairs, cibarium teeth and spermathecae. The abdominal hairs were used to differentiate between Phlebotomus and Sergentomyia species. S. gemmea and S. hivernus had similar cibarium hind teeth but they were distinguished clearly by the morphological characteristics of their spermathecae. Recently, Phumee et al. (2016) have distinguished S. hivernus and S. iyengari by the morphology of cibarial teeth and spermathecae. The cibarium of S. hivernus contains 13-14 hind teeth and one row of fore teeth and the spermathecae are smooth, long and tubular without obvious junctions between the body and duct. S. iyengari has a cibarium which contains 13-14 hind teeth, the central teeth are smaller than the rest, and fore teeth are absent. Its spermathecae are shorter with 7	  	  easily observed junctions between the body and duct. The morphological characteristics of spermathecae of S. hivernus collected in this current study correspond with the report of Phumee et al. (2016). S. gemmea was the most abundant species in the area. This statistic is similar to that in previous studies in the south of Thailand, i.e., Phang-nga, Suratthani, Nakonsitammarat, Songkhla (Sukmee et al., 2008; Sukra et al., 2013; Chusri et al., 2014), and Trang (Kanjanopas et al., 2013). The other abundant species in the study area were P. stantoni and S. barraudi. It has been suggested that S. gemmea and S. barraudi might be possible vectors of the leishmaniasis in this area since Chusri et al. (2014) have detected Leishmania DNAs in both species. In addition, Phumee et al. (2016) have detected unknown Trypanosoma DNA in P. stantoni collected from southern Thailand. Although investigation of Leishmania or Trypanosoma parasites and DNA in unidentified sand flies in this study area has been performed in our laboratory, we have yet not found any parasites and/or DNA in the flies (unpublished data). More investigation of the vector status of S. gemmea and S. barraudi is required in all infected areas in Thailand. Furthermore, L. martiniquensis and “L. siamensis” have been reported as members of the Leishmania enriettii complex (Bualert et al., 2012; Leelayoova et al., 2013; Kwakye-Nuako et al., 2015). Dougall et al. (2011) have presented strong evidence that the biting midge Forcipomyia (Lasiohelea) sp.1 and possibly Forcipomyia (Lasiohelea) peregrinator may be transmitting Australian Leishmania, a member of the L. enriettii complex, in northern Australia. Seblova et al. (2015) have demonstrated that L. enriettii could develop late stage infection in the biting midge Culicoides sonorensis and that the late stage parasites could produce large, ulcerated, tumour-like lesions in hamsters. The studies have supported the hypothesis that some biting midges could be natural vectors of the L. enriettii complex. 8	  	  Therefore, investigation of the vector status of midges is required in all infected areas in Thailand. Previous studies in many caves in Thailand have shown that S. gemmea has been a less common species (~0.5%) (Polseela et al., 2007, 2011; Apiwathnasorn et al., 2011). Caves in Thailand, generally, have low temperatures, around 24.4 to 32.3°C, with relative humidity around 64 to 95% (Polseela et al., 2007; Polseela et al., 2011; Apiwathnasorn et al., 2011). The distribution information from our current study and the studies in southern Thailand suggest that S. gemmea is likely to be more successful in slightly warm and humid habitats near human living areas. In conclusion, five species of sand flies collected from the Leishmania-infected area in Hang Dong, Chiang Mai, Thailand were identified. S. gemmea and S. hivernus were found in Chiang Mai for the first time. Also, S. gemmea was the most abundant species in the area. This finding provided information for further studies on the vector status of these sand fly species in Thailand.  Acknowledgements. This study was supported by a Diamond Research Grant of the Faculty of Medicine, Chiang Mai University to Pradya Somboon. In addition we acknowledge the Research Administration Office of Chiang Mai University which provided the budget for our Excellence Center in Insect Vector Study. The work was partially funded by the Korean International Cooperation for Infectious Diseases (KOICID). We thank Chiang Mai University for the award of the Post-Doctoral Fellowship 2016 to Pradya Somboon and Sriwatrapron Sor-suwan and the Newton Fund-TRF Travel Grant to PhD Supervisor Year 2014/15 to Narissara Jariyapan.  Conflict of Interest 9	  	  The authors declare that they have no conflict of interest.  REFERENCES Apiwathnasorn, C., Samung, Y., Prummongkol, S., Phayakaphon, A. & Panasopolkul, C. (2011). Cavernicolous species of phlebotomine sand flies from Kanchanaburi Province, with an updated species list for Thailand. Southeast Asian Journal of Tropical Medicine and Public Health, 42: 1405-1409. Bualert, L., Charungkiattikul, W., Thongsuksai, P., Mungthin, M., Siripattanapipong, S., Khositnithikul, R., Naaglor, T., Ravel, C., El Baidouri, F. & Leelayoova, S. (2012). Autochthonous disseminated dermal and visceral leishmaniasis in an AIDS patient, southern Thailand, caused by Leishmania siamensis. American Journal of Tropical Medicine and Hygiene, 86: 821-824. Chiewchanvit, S., Tovanabutra, N., Jariyapan, N., Bates, M.D., Mahanupab, P., Chuamanochan, M., Tantiworawit, A. & Bates, P.A. (2015). Chronic generalized fibrotic skin lesions from disseminated leishmaniasis caused by Leishmania martiniquensis in two patients from northern Thailand infected with HIV. British Journal of Dermatology, 173: 663-670. Chusri, S., Thammapalo, S., Silpapojakul, K. & Siriyasatien, P. (2014). Animal reservoirs and potential vectors of Leishmania siamensis in southern Thailand. Southeast Asian Journal of Tropical Medicine and Public Health, 45: 13-19. Dougall, A.M., Alexander, B., Holt, D.C., Harris, T., Sultan, A.H., Bates, P.A., Rose, K. &Walton, S.F. (2011). Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia. International Journal for Parasitology, 41: 571-579. 10	  	  Kanjanopas, K., Siripattanapipong, S., Ninsaeng, U., Hitakarun, A., Jitkaew, S., Kaewtaphaya, P., Tan-ariya, P., Mungthin, M., Charoenwong, C. & Leelayoova, S. (2013). Sergentomyia (Neophlebotomus) gemmea, a potential vector of Leishmania siamensis in southern Thailand. BMC Infectious Diseases, 13: 333. Kwakye-Nuako, G., Mosore, M.T., Duplessis, C., Bates, M.D., Puplampu, N., Mensah-Attipoe, I., Desewu, K., Afegbe, G., Asmah, R.H., Jamjoom, M.B., Ayeh-Kumi, P.F., Boakye, D.A. & Bates, P.A. (2015). First isolation of a new species of Leishmania responsible for human cutaneous leishmaniasis in Ghana and classification in the Leishmania enriettii complex. International Journal for Parasitology, 45: 679-684. Leelayoova, S., Siripattanapipong, S., Hitakarun, A., Kato, H., Tan-ariya, P., Siriyasatien, P., Osatakul, S. & Mungthin, M. (2013). Multilocus characterization and phylogenetic analysis of Leishmania siamensis isolated from autochthonous visceral leishmaniasis cases, southern Thailand. BMC Microbiology, 13: 60. Lewis, D.J. (1978). The Phlebotomine sandflies (Diptera: Psychodidae) of the Oriental Region. Bulletin of the British Museum (Natural History) Entomology, 37: 217-343. Lewis, D.J. (1987). Phlebotomine sandflies (Diptera: Psychodidae) from the Oriental Region. Systematic Entomology, 12: 163-180. Phumee, A., Tawatsin, A., Thavara, U., Pengsakul, T., Thammapalo, S., Depaquit, J., Gay, F. & Siriyasatien, P. (2016). Detection of an unknown Trypanosoma DNA in a Phlebotomus stantoni (Diptera: Psychodidae) collected from southern Thailand and records of new sand flies with reinstatement of Sergentomyia hivernus Raynal & Gaschen, 1935 (Diptera: Psychodidae). Journal of Medical Entomology, doi:10.1093/jme/tjw161  11	  	  Polseela, R., Apiwathnasorn, C. & Samung, Y. (2007). Seasonal variation of cave-dwelling phlebotomine sandflies (Diptera:Psychodidae) in Phra Phothisat Cave, Saraburi Province, Thailand. Southeast Asian Journal of Tropical Medicine and Public Health, 38: 1011-1015. Polseela, R. (2012). Sand fly and emerging disease: Leishmaniasis. Thammasat Medical Journal, 1: 77-96. Polseela, R., Vitta, A., Nateeworanart, S. & Apiwathnasorn, C. (2011). Distribution of cave-dwelling phlebotomine sand flies and their nocturnal and diurnal activity in Phitsanulok Province, Thailand. Southeast Asian Journal of Tropical Medicine and Public Health, 42: 1395-1404. Polseela, R., Depaquit, J. & Apiwathnasorn, C. (2016a). Description of Sergentomyia phadangensis n. sp. (Diptera, Psychodidae) of Thailand. Parasites and Vectors, 9: 21. Polseela, R., Jaturas, N., Thanwisai, A., Sing, K.W. & Wilson, J.J. (2016b). Towards monitoring the sandflies (Diptera: Psychodidae) of Thailand: DNA barcoding the sandflies of Wihan Cave, Uttaradit. Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis, 27: 3795-3801. Quate, L.W. (1962). A review of the Indo-Chinese Phlebotomine (Diptera: Psychodidae). Pacific Insects, 4: 251-267. Seblova, V., Sadlova, J., Vojtkova, B., Votypka, J., Carpenter, S., Bates, P.A. & Volf, P. (2015). The Biting Midge Culicoides sonorensis (Diptera: Ceratopogonidae) Is Capable of Developing Late Stage Infections of Leishmania enriettii. PLoS Neglected Tropical Diseases, 9: e0004060. Sukmee, T., Siripattanapipong, S., Mungthin, M., Worapong, J., Rangsin, R., Samung, Y., Kongkaew, W., Bumrungsana, K., Chanachai, K., Apiwathnasorn, C., 12	  	  Rujirojindakul, P., Wattanasri, S., Ungchusak, K. & Leelayoova, S. (2008). A suspected new species of Leishmania, the causative agent of visceral leishmaniasis in a Thai patient. International Journal for Parasitology, 38: 617-622. Sukra, K., Kanjanopas, K., Amsakul, S., Rittaton, V., Mungthin, M. & Leelayoova, S. (2013). A survey of sandflies in the affected areas of leishmaniasis, southern Thailand. Parasitology Research 112: 297-302. Theodor, O. (1938). On Sandflies (Phlebotomus) from Ceylon, Siam, and Malay. Indian Journal of Medical Research, 26: 261-269. World Health Organization (WHO). (2016). Leishmaniasis, fact sheet Updated March 2016. (http://www.who.int/mediacentre/factsheets/fs375/en/)   13	  	  Figure legends   Figure 1. Map of Chiang Mai, northern Thailand showing the geographic location where sand flies were collected.   14	  	     Figure 2. Morphological characteristics of cibarium teeth and pigment patches of female sand flies. (a) S. gemmea (b) S. hivernus (c) S. indica (d) S. barraudi (e) P. stantoni.   15	  	     Figure 3. Morphological characteristics of spermatheca of female sand flies (a) S. gemmea (b) S. hivernus (c) S. indica (d) S. barraudi (e) P. stantoni.   16	  	     Figure 4. Relationship between diversity of collected sand flies and the climate parameters of relative humidity and temperature in the Leishmania infected area from January to August 2016.   17	  	  Table 1. Species identification numbers of 280 female sand flies in this study from January to August 2016 Species Month Percentage Jan Feb Mar Apr May Jun Jul Aug Total P. stantoni 3 5 4 7 15 23 5 17 79 28.22 S. gemmea 9 9 5 13 6 24 20 13 99 35.36 S. barraudi 2 2 2 4 6 11 10 15 52 18.57 S. indica 0 2 7 9 3 21 3 3 48 17.14 S. hivernus 0 0 1 0 0 0 1 0 2 0.71 Total 14 18 19 33 30 79 39 48 280 100   

Species composition and population dynamics of phlebotomine sand flies in a Leishmania infected area of Chiang Mai, Thailand

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Species composition and population dynamics of phlebotomine sand flies in a Leishmania infected area of Chiang Mai, Thailand

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