In Limestone Caves, Khao Pathawi, Uthai Thani Province, Thailand

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Distribution of Phlebotomine Sand Flies in Limestone Caves

DISTRIBUTION OF PHLEBOTOMINE SAND FLIES (DIPTERA:PSYCHODIDAE) IN LIMESTONE CAVES, KHAO PATHAWI, UTHAI THANI PROVINCE, THAILAND

R Polseela1,2, A Vitta 1,2 and C Apiwathnasorn3

1Department of Microbiology and Parasitology, 2Centre of Excellence in Medical Biotechnology, Faculty of Medical Science, Naresuan University, Phitsanulok, 3Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

Abstract. This study investigated the species composition and density of the sand flies found inside four limestone caves at Khao Pathawi, Thap Than District, Uthai Thani Province. Sand flies were collected using Centers for Disease Control (CDC) light traps from October 2012 to September 2013. The sand flies were captured between 06:00 pm - 06:00 am. A total of 11,817 sand flies were collected with a male:female ratio of 1.0:1.2 (5,325:6,492). The specimens were identified as eight species belonging to three genera Phlebotomus, Sergentomyia, Chinius, and com- prised of S. anodontis, P. argentipes, P. stantoni, S. barraudi, S. silvatica , S. gemmea, S. indica, and C. barbazani. Sergentomyia anodontis (55.0%) was the predominant species followed by P. argentipes (33.6%) and others. Five species of sand fly were found throughout the year in this area: P. argentipes, P. stantoni, S. anodontis, S. barraudi and S. gemmea. The highest average density of sand flies was found in Ratree cave (35.0 sand flies per trap per night) and lowest in Bandai cave (29.0 sand flies per trap per night). The population of sand fly fluctuated from the highest peak in December (28.5%) to the lowest peak in May (2.3%). The distribution of sand fly species in attraction areas is important for the control program of infection risk of leishmaniasis. Keywords: sand flies, CDC light traps, leishmaniasis, limestone cave, Thailand

INTRODUCTION larly members of genera Lutzomyia and Phlebotomus. Phlebotomine sand flies Leishmaniasis is a vector-borne dis- (Diptera:Psychodidae) are found in a wide ease caused by the protozoan parasite range of habitats. The genus Phlebotomus of Leishmania species. The parasite is is responsible for the transmission of transmitted by female sand flies, particu- leishmaniasis in the Old World. About Correspondence: R Polseela, Department of Mi- 40 species of Phlebotomus spp in the Old crobiology and Parasitology, Faculty of Medi- World have been proven or suspected to cal Science, Naresuan University, Phitsanulok be vectors of Leishmania spp (Alexander 650000, Thailand. and Maroli, 2003). Leishmania spp have a E-mail: [email protected],polseela@hotmail. wide geographic distribution. Some spe- com cies of sand flies are adapted to a range

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by Leishmania donovani, L. infantum and L. siamensis (Kong- kaew et al, 2007; Maharom et al, 2008; Bualert et al, 2012). L. siamensis is a new species found in Thailand where it is the cause of visceral leishmaniasis (Sukmee et al, 2008; Bualert et al, 2012). P. argentipes is the species of sand flies involved in the transmission of leishmaniasis and it can be found in Thai- land. At present, Sergentomyia gemmea may be a potential vec- Fig 1–Outside a limestone cave at Khao Pathawi, Thap Than tor of L. siamensis which is the District, Uthai Thani Province. cause of visceral leishmaniasis in Thailand (Kanjanopas et al, 2013). The sand flies fauna is presented in different loca- tions such as cave, forest, rural, urban, sylvatic and domestic areas (Magill et al, 1993). Previ- ous studies have shown a di- versity of sand flies in different parts of Thailand. Cave-dwelling phlebotomine sand flies are represented by different spe- Fig 1–Outside limestone caves in Khao Pattawee, Thap Than cies in different sites (Polseela District, Uthai Thani Province. et al, 2007; 2011a,b; Shahar et al, Fig 2–Inside limestone caves in Khao Pattawee, Thap Than, 2011). Uthai Thani Province. The objective of this study was to determine the species Fig 2–Inside a limestone cave at Khao Pathawi, Thap Than, composition and seasonal District, Uthai Thani Province. fluctuations of sand fly species inside caves within a of environmental conditions with a wide single mountain region. This may assist distribution. About 30 species of sand flies in planning for programs of prevention are proven vectors of at least 20 Leishmania and control of leishmaniasis. species (Emamia and Yazdib, 2008). In Thailand, there are 5 genera (Phlebo- MATERIALS AND METHODS tomus, Sergentomyia, Idiophlebotomus, Chin- ius and Nemopalpus) including 27 species of Study area sand flies reported from different habitats The study collected sand flies from (Apiwathanasorn et al, 1989; 2011). Autoch- October 2012 to September 2013 from thonous cases of leishmaniasis are caused four limestone caves in Khao Pathawi,

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Thap Than District, Uthai Thani Province: flies were captured from the four caves. Ratree cave, Bandai cave, Aunag cave, The highest peak of activity was found and Phuttha Prawat cave. These caves during December and then decreased to situated on mountainsides at 15°28’40.1”N, the lowest point in May (Tables 1 and 2). 099°45’54.7”E (Figs 1 and 2). All caves are Eight species were identified of which about 10-50 m wide, 15-50 m long and 5 belonged to the genus Sergentomyia, 2 5-30 m high. There are some birds and bats to the genus Phlebotomus, 1 to the genus inside caves. The caves are surrounded by Chinius (Table 2). The greatest number green trees and cover mainly by forest veg- of specimens was collected in December, etation. The caves are near a village. In this while P. argentipes, P. stantoni, S. barraudi, area, measures of temperature and relative and S. anodontis were trapped through- humidity were recorded monthly with a out the whole year (Table 2). Both male digital hygro-thermometer. The average and female flies were attracted by the temperature during the study were 25.0- light. The male to female ratio was 1.0:1.2 32.4°C. The annual average relative humid- (6,492:5,325). The average density was ity is 54-94%. Rainfall data were provided 29.03-35.05 sand flies per light trap per by the Thai Meteorological Department. night. The highest average density was Sand fly collection and identification found in Ratree cave (35.05 sand flies per trap per night) and the lowest in Bandai A survey of sand flies population was cave (29.03 sand flies per trap per night). made using 32 miniature CDC light traps The known vector, P. argentipes had its one night per month (Sudia and Cham- peak in December (Fig 3). The monthly berin,1962). CDC light traps were placed variation of sand fly density is presented inside the cave at 5-8 m from each loca- in Fig 4. The minimum and maximum tion. The trap were set near crack of rock. temperature and relative humidity values The traps were operated continuously ranged between 22.5-34.5ºC, and 54-94%, for 12 hours (06:00 pm to 06:00 am) and respectively. the insects were collected after 12 hours period. During each month of survey in these caves, the sand flies were captured DISCUSSION at the same site. A survey of sand fly activity was The sand flies were separated from conducted for a 12 month period inside other arthropods under a stereo-micro- four limestone caves using CDC light scope and stored in 80% alcohol for identi- traps. The nocturnal activity among the fication. Head and thorax of sand flies were different species of sand flies was studied separated and mounted with Hoyer’s me- (Apiwathnasorn et al, 1989, 1993; Polseela dium on glass slides. Taxonomic keys were et al, 2007, 2011). The greatest numbers of used for identifications base on the mor- specimens were collected in December. phology of female spermatheca, pharyngx, In this study, 8 species were identified: and male genitalia (Lewis, 1978). P.argentipes, P. stantoni, Sergentomyia anodontis, S. barraudi, S. silvatica, S. gem- RESULTS mea, S. indica, and Chinius barbazani. In Thailand, previous captures of sand flies Sand fly species composition and sex ratio documented 27 species (Apiwathnasorn A total of 11,817 phlebotomine sand et al, 1989; 1993; 2011). There are some

Vol 46 No. 3 May 2015 427 Southeast Asian J Trop Med Public Health % 30.5 45.1 54.9 29.5 18.6 21.4 100.0

100 Total 1,011 1,245 1,632 1,852 1,437 2,170 3,607 1,191 1,279 5,325 6,492 3,484 2,202 2,524 11,817 1.0:1.2 13 49 63 12 34 21 46 34 6.1 Sep 112 321 210 531 395 328 723 76 Aug 160 129 145 62 61 41 52 52 93 212 39 30 33 99 72 31 27 39 49 75 84 70 Jul 3.1 4.8 173 170 199 392 372 562 65 39 42 81 27 48 75 52 53 37 3.1 Jun 183 180 363 65 76 28 105 26 102 137 2013 May 7 15 14 10 91 52 38 53 38 18 13 15 16 25 29 3.0 2.3 109 74 200 139 105 193 135 166 134 359 269 Apr Table 1 Table 30 26 28 54 17 18 35 43 73 3.6 116 142 189 231 420 258 Mar 13 57 36 93 58 32 90 58 19 18 77 31 2.5 Feb 186 105 291 66 Jan 118 113 248 280 528 248 290 538 144 680 827 179 262 12.8 1,507 602 245 301 546 286 544 830 380 506 509 Dec 28.5 Uthai Thani Province from October 2012 to September 2013. September to 2012 October from Province Thani Uthai 1,111 1,513 1,860 3,373 113 147 483 440 923 152 410 562 148 261 886 120 267 930 17.0 2012 Nov 1,083 2,013

90 110 209 294 503 169 395 564 101 191 197 307 578 987 Oct 13.2 1,565 The number of sand flies captured by CDC light traps in limestone caves at Khao Pathawi, Thap Than District, District, Than Thap Pathawi, Khao at caves limestone in traps light CDC by captured flies sand of number The Male Female Total Male Female Total Male Female Total Female Male Total Female Male Total % Male:Female Cave Bandai Phuttha Prawat Aunag Ratree Total

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1,200 December. The greatest number of specimens was collected from 1,000 a location near rock fissure in the cave. The density variation of 800 sand fly observed in this study may indicate the present of sand 600 flies to survive in the different

400 caves. Sand flies in Phra Photh- Number of sand files isat cave in Saraburi Province had

200 their highest peak in July (436 sand flies per light trap per night)

0 (Polseela et al, 2007). Currently there are two sand fly vectors of Month visceral leishmaniasis in other Fig 3–Nocturnal activity of Phlebotomus argentipes cap- countries known in Thailand: tured by CDC light traps in limestone caves at Khao P. argentipes and P. major major Pathawi, Thap Than, Uthai Thani Province from (Swaminath et al, 2006; Azizi et al, October 2012 to September 2013. 2008). Of these, we found P. argentipes sand fly in this study. Thus 120 a clear demarcation of the geographic limits of sand fly species 100 and activity was demonstrated, according to the type of cave and 80 different environments of this area. The findings also indicate 60 a health risk for both locals and

40 visitors to the caves in this area. Our previous study in Phra

Number of sand files per trap night 20 Phothisat cave and Tham Phra Pho Thisat Temple that had their 0 highest peak in July and March Month (Polseela et al, 2007, 2011a). Thir- Fig 4–Monthly variation of sand fly density captured by teen species were found in Phra CDC light traps in limestone caves at Khao Pathawi, Phothisat cave, and 16 species Thap Than, Uthai Thani Province from October 2012 were found in Tham Phra Pho to September 2013. Thisat Temple, Saraburi Province. There are some differences in the species composition found in species present in this area and others difference area. P. argentipes was found at have disappeared, perhaps due to differ- the level of 0.8-2.2% in Saraburi Province ent environmental aspects in each area. S. and 20.2% in Naresuan cave, Phitsanulok barraudi was the most abundant species (Polseela et al, 2007; 2011a, b). In northern (55.0%). P. argentipes, a proven vector of Israel, sand fly populations were preva- Leishmania donovani, accounted for 33.6%. lent from April to November and peaked P. argentipes showed its highest peak in between June and August (Kravchenko

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et al, 2004). In January and February 2006, the sand fly was apparently ab- - (%) (5.0) (1.2) (100.0) (55.0) (2.2) (33.6) (0.1) (0.3) (2.7) sent in Saudi Arabia (Badry et al, 2008). 14 31 597 141 256 314 Coleman et al (2006) reported sand Total 6,493 3,971 11,817

(100.0) fly activity periods in Iraq with the highest mean monthly captured rates in August, 2003 and July, 2004. Sand 0 0 36 12 37 21 Sep 364 723 253 flies were active especially during the (6.1) dry season (from May to November) 0 0 in Marrakech, Morocco. The maximum 16 12 30 Aug 196 of species richness was in May, when the daily temperature ranged between 4 27 11

Jul 13ºC and 26ºC. The species differed in 194 281 372 562 122 (3.2) (4.8) their activity period; P. sergenti, was active especially during April, May 9 8 20 29

Jun and June (Boussaa et al, 2005). These 188 363 105 (3.1) seasonal patterns should be related to different environmental factors. Noc- 7 5 0 7 9 0 8 24 14 6 91 2013 May turnal activity of sand flies may result in transmitted pathogens to animals 8 0 4 and humans. Those people living near 23 13 12 19 118 175 130 359 269 Apr

(3.0) (2.3) the cave may be particularly at risk of exposure to such pathogens. 5 0 0 9 Table 2 Table 16 12 Both sexes of the sand fly were 240 420 138 Mar (3.6) found to have nocturnal activity in this study. The male to female ratio 2 0 0 6 3 11 Feb 145 291 124 was 1.0:1.2. This suggests that both (2.5) sexes of the sand fly are attracted to 0 2 0 the light traps. Previous studies have 58 30 15 Jan 893 509 shown the number of male sand flies 1,507 (12.7) captured using CDC light traps was significantly higher than that of female 47 113 165

Dec with a male:female ratio of 2.5:1 and Uthai Thani Province from October 2012 to September 2013. September to 2012 October from Province Thani Uthai 1,113 1,867 3,373 (28.5) 1.9:1 in Saraburi, Thailand (Polseela

0 et al, 2007; 2011a). According to a study 89 10 12 6 16 25 62 674

2012 conducted in Cukurova plain, Tur- Nov 1,187 2,013 (17.0) key, males were found to be 4.7 times 0 0 more abundant than females (Kasap 21 31 37 119 829 528 Oct et al, 2009). In different areas, the 1,565 (13.2) number of males captured was higher

than that of females, with the sex

ratio of 2.3:1 reported by Boussaa et al (2005), 3.4:1 by Hanafiet al (2007),

The monthly distribution of sand flies species captured by CDC light in limestone caves at Khao Pathawi, Thap Than, Than, Thap Pathawi, Khao at caves limestone in light CDC by captured species flies sand of distribution monthly The 1.12:1 by Badry et al (2008) and 2:1 Species S.anodontis P.argentipes S.barraudi S.gemmea P.stantoni S.silvatica S.indica C.barbazani Total (%) by Emamia and Yazdib (2008). The

430 Vol 46 No. 3 May 2015 Distribution of Phlebotomine Sand Flies in Limestone Caves higher proportion of males in the captures thonous human cases in Thailand and on with CDC light traps could be explained the presence of the Leishmania siamensis, by the natural behavior of the males to it is possible that S.gemmea phlebotomine follow the females to assure fertilization sand fly might be involved in the cycle during their displacements (Barata et al, of leishmaniasis in Thailand. The study 2004). Male sand flies have a weaker dis- of population activity pattern might be persal tendency than females and limit helpful in the implementation of success- their flight to questing for food plants ful tactics for vector control and disease (Yuval and Schlein, 1986; Janini et al, prevention. 1995). The hopping behavior leads to the assumption that they do not disperse far ACKNOWLEDGEMENTS from the breeding site (Killick-Kendrick, 1999). However, only the female sand fly This study was conducted under the can feed on blood and be the vector for financial support of Naresuan Univer- leishmaniasis. sity, Phitsanulok Province, Thailand. We For this study’s month-by-month col- would like to thank the staff at Khao Pat- lection, the highest peak of capture was tawee, Thap Than District, Uthai Thani in December, and lowest in May. Tem- Province for their kind cooperation during perature and humidity are factors closely the field study; Mr Banjong Deelua and related to duration of metamorphosis staff for the collection of specimens; and (Theodor, 1936). However, the optimum Professor Gavin Reynolds for assistance rearing temperatures differ among spe- with the editing of this paper. cies of sand flies. Guernaoui et al (2006) reported that variations in the nocturnal REFERENCES activity rhythm are related to variations in temperature and relative humidity. The Alexander B, Maroli M. Control of Phleboto- most favorable temperatures for devel- mine sand flies. Med Vet Entomol 2003; 17: 1-18. opment of sand flies are between 25 ºC and 30ºC (Safyanova, 1964). The activity Apiwathnasorn C, Sucharit S, Rongsriyam Y, of sand flies is continuous through the et al. A brief survey of Phlebotominae sand Southeast Asian J Trop Med night and that relative humidity has a flies in Thailand. Public Health 1989; 20: 429-32. significant effect on the nocturnal activity. However, sand fly activity was closely Apiwathnasorn C, Sucharit S, Surathi K, Deesin related to climatic factors. Many investi- T. Anthropophilic and zoophilic phlebotomine sand flies (Diptera:Psychodidae) gations have demonstrated the existence from Thailand. J Am Mosq Control Assoc of seasonal variations in the nocturnal 1993; 9: 135-7. activity of sand flies (Merrisonet al, 1995; Apiwathnasorn C, Samung Y, Prummongkol Polseela et al, 2011a). S, Phayakaphon A, Panasopolkul C. According to our findings, sand Cavernicolous species of Phlebotomine flies are active in the different caves. The sand flies from Kanchanaburi province, high number of flies are found in several with an update species list for Thailand. sites in the cave. The continental climatic Southeast Asian J Trop Med Public Health environment inside the cave provides 2011; 42: 1405-9. a possible breeding site of the potential Azizi K, Rassi Y, Javadian E, et al. First detec- vector. Based on the occurrence of autoch- tion of Leishmania infantum in Phlebotomus

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