J Arthropod-Borne Dis, December 2019, 13(4): 399–406 K Arzamani et al.: Comparative Performance of … 399 http://jad.tums.ac.ir Published Online: December 31, 2019 Original Article Comparative Performance of Different Traps for Collection of Phlebotominae Sand Flies and Estimation of Biodiversity Indices in Three Endemic Leishmaniasis Foci in North Khorasan Province, Northeast of Iran Kourosh Arzamani1; *Yavar Rassi2; *Hassan Vatandoost2,3; Amir Ahmad Akhavan2; Mohammad Reza Abai2; Mohammad Alavinia1,4; Kamran Akbarzadeh2; Mehdi Mohebali5; Sayena Rafizadeh6; Fateh Karimian2; Mehdi Badakhshan2; Azad Absavaran2 1Vector-Borne Diseases Research Center, North Khorasan University of Medical Sciences, Bojnurd, Iran 2Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Department of Environmental Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 4Toronto Rehabilitation Centre, University Health Network, Toronto, Canada 5Department of Medical Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 6Ministry of Health and Medical Education, Tehran, Iran (Received 04 Aug 2019; accepted 30 Dec 2019) Abstract Background: Phlebotominae sand flies (Diptera: Psychodidae) are the vectors of leishmaniasis. There are different methods for sand fly collection with different performance. The purpose of the current study was to compare the effect of different traps for collection of Phlebotominae sand flies in three endemic leishmaniasis foci in North Khorasan Prov- ince, northeast of Iran. Methods: Sand flies were collected using seven different traps from three villages, three times each twenty days during peak periods of seasonal activity in 2016. Results: A total of 7253 sand flies were collected. The specimens belonged to19 species. Phlebotomus sergenti was the most predominant species in the study area. Light trap baited with Carbon dioxide (CLT) and sticky paper trap (SPT) caught 22.6% and 22.3% of sand flies respectively. Animal baited trap (ABT) and white Shannon trap (WST) caught significantly fewer sand flies than the other traps. The sex ratio was different by phlebotominae sand fly species and collection methods. The sex ratio was highest in SPT and lowest in black Shannon trap (BST). Species diversity and species richness in SPT were more than other traps. Conclusion: Our findings confirm that CLT and SPT are the most efficient sand fly collection methods. CLT is higher attractive for females and Phlebotomus genus and is an ideal method for monitoring the population of Phlebotomus ge - nus during surveillance. SPT is an inexpensive, convenient and easy to be used to detect the presence of sand flies at low densities and provide a more realistic estimation of sand flies biodiversity. Keywords: Collection method; Biodiversity; Sandflies; Leishmania; Iran Introduction Leishmaniasis is transmitted to human in 98 countries and 3 territories on 5 continents in the world. There are three main forms of leish- maniases, visceral leishmaniasis (VL), cutane- ous leishmaniasis (CL), and mucocutaneous leishmaniasis (1). In Iran, VL and CL are endemic and this country is one of the ten countries with the highest estimated cases of CL. In the North Khorasan Province VL and CL are endemic. Leishmania infantum have been detected in reservoir and vector of the disease. Numerous cases of VL have been recorded during the last decades in humans. About 160 confirmed *Corresponding authors: Dr Yavar Rassi, E-mail: rassi184@gmail.com, Dr Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com http://jad.tums.ac.ir/ mailto:rassi184@gmail.com mailto:hvatandoost1@yahoo.com J Arthropod-Borne Dis, December 2019, 13(4): 399–406 K Arzamani et al.: Comparative Performance of … 400 http://jad.tums.ac.ir Published Online: December 31, 2019 human cases of VL have been diagnosed and registered especially from the north half of the province during the period 1990–2010. During 2005 to 2008, about 1453 and between the years 2006–2013 at least 2831 patients with CL were reported from different districts of the prov- ince (2-7) . Phlebotominae sand flies (Diptera: Psycho- didae) are the vectors of leishmaniasis and some human pathogens. There are approximately 1,000 valid described species of sand flies in the world (8). The first step for study of Phlebotominae sand flies, their behavior, taxonomy, ecology or determination of infection is collection of them. There are several methods and traps for collection of phlebotominae sand flies (9). A variety of phlebotominae sand flies collection methods have previously been studied in dif- ferent countries. In a study in Turkey among different traps, light trap baited with Carbon dioxide (CLT) were found to be more stable and productive method than others trap for both estimating the species composition and the pop- ulation density of sand flies in the study area (10). In an investigation in Morocco statistical analysis showed that significantly more num- ber of sand flies were obtained using sticky paper traps (SPT) compared with light traps (LTP), therefore SPT was more effective than LTP (11). In northeastern of Italy three standard meth- ods for collection of sand flies (SPT, LTP, and CLT) were compared. CLT were more attrac- tive for females of P. perniciosus and P. ne- glectus. LTP showed an intermediate efficien- cy and were more attractive for P. neglectus, compared to other two traps. Results suggest that in northern Italy the CLT is a suitable sampling method for sand fly monitoring pro- grams (12). In center of Iran eight methods for collec- tion of sand flies were compared and 37.3% of all phlebotominae sand flies were collected using Disney trap (DST). These proportions were 24.2%, 10.5%, 8.5% and7.3% in SPT, Black Shannon Traps (BST), Animal Baited Trap (ABT) and White Shannon Traps (WST) respectively. CLT (5.0%), LTP (3.3%) and Ma- laise trap (0.1%) showed a low efficiency (13). In Brazil to compare the relative attrac- tiveness of BST and WST for sand flies, sev- eral pairs of traps were placed side by side in front of caves in four areas. The result revealed that BST was much more productive than the WST, especially for anthropophilic species (14). According to the previous study tolerance to DDT reported in North Khorasan Province, therefore collection and monitoring of phlebotominae sand flies is necessary (15). There is no sufficient document for evaluation of different collection methods of phlebotom- inae sand flies in Iran. The purpose of this study was to compare performance of different traps in three endemic leishmaniasis foci in North Khorasan Province, northeast of Iran to assess which of these traps could catch a large num- ber or show the most diversity of sand flies. Materials and Methods Study area This cross-sectional study was conducted during 2016 in North Khorasan Province, be- tween 36°37′-38°17′ N latitudes and 55°53′- 58°20′ E longitudes. The province has a desert, mountain and temperate climate with cold winters and receives about 250mm of rainfall annually. The total area was approximately 28,434km2. The province is bordered by Turk- menistan in the North and situated in northeast of Iran. Bojnurd is the capital city of the prov- ince (Fig. 1). Selection of study villages Three villages in the province were select- ed, where human VL and/or CL had been re- ported in the last 5 years. Bacheh-Dareh vil- lage (A): It is located in Raz and Jargalan Coun- ty, 15km from the border of Turkmenistan. Sev- eral human VL and CL cases had been report- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 399–406 K Arzamani et al.: Comparative Performance of … 401 http://jad.tums.ac.ir Published Online: December 31, 2019 ed in the last 5 years. Collection site was a valley 3km far from the village and sand flies captures have been restricted to outdoor and wild environments. Kohne-Jolgeh village (B): It is located in Maneh and Samalqan County. Several human VL and CL had been reported in the last 5 years. Collection site was indoors and domestic environments. Arg village (C): It is located in Jajarm County. Only human CL cases had been reported from this village. Col- lection site was indoors and outdoors and do- mestic environments. Sand fly collection Sand flies were collected for three times each twenty days during the peak periods of seasonal activity. Sample collection began from early Jul and continued until late Aug in 2016. Sand flies were collected using seven different traps including: 1. Sticky paper Traps (SPT) consisting of a white paper sheet with size of 15×21cm that coated with castor oil. We used 10 paper traps for each rotation and totally 60 papers per night. 2. CDC Light Trap (LTP). The light traps pow- ered by alternating current and were suspend- ed at 1.5 meters above the ground. 3. CDC light trap baited with Carbon dioxide (Co2) gas (CLT) that obtained by a Co2 gas tank. 4. White Shannon Trap (WST) was made of white cloth and consisted of a large central compartment and two smaller lateral ones. The measurements, (width, length and height), of the central and the lateral compartments were, respectively: 1.3×1.3×2 meters and sus- pended by cords from supports and base of the traps touched the ground. A portable stove was used as Co2 and light source inside the traps. 5. Black Shannon Trap (BST). This trap was similar to WST except that were made of black cloth. 6. Disney Trap (DST). A cage, holding the chicken bait in the middle and surrounded by sticky paper traps. 7. Animal Baited Trap (ABT). A net trap (2×3×2 meters) was used to sample sand flies at- tracted to a chicken as animal bait (Fig. 2). All of the traps were placed randomly with a distance of about 20 meters of each other. The traps were set before sunset, were changed every two hours and remained in operation during the night (20:00 to 08:00). As the traps were changed, the new ones were replaced in the same location. Traps were rotated clock- wise between the trap locations in site “A” and “C” but were fix in site “B”. Collected sand flies were stored in 96% ethanol alcohol. The specimens were mounted on glass slides in Puri’s medium. Species identification was car- ried out according to morphological characters with several taxonomic keys of sand flies (16- 17). Statistical analyses The statistical analyses were performed in SPSS program. We evaluated the comparative performance of different traps for each collec- tion site. Shannon-Wiener index have been calculated to estimate species biodiversity of Phlebotomine sand flies in different traps (18- 19). Ethical statement Specimen collection was performed in ac- cordance with the procedures approved by the Ethical Committee of North Khorasan Uni- versity of Medical Sciences. Results A total of 7253 sand flies were collected. Frequency, gender and percent of Phlebotom- ine sand flies collected by different traps in three collection sites are shown in Table 1. On collection site A we collected the most abun- dance of sand flies and in collection site C the fewer of specimens collected. The specimens were belonged to19 species, 10 species of the genus Phlebotomus and 9 of the genus Sergentomyia. In A collection site, we collected 18 species and more sand fly spe- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 399–406 K Arzamani et al.: Comparative Performance of … 402 http://jad.tums.ac.ir Published Online: December 31, 2019 cies collection appears in this area. Significant- ly fewer sand flies collected in site C with A collection of only four species and in site B eight species were caught. Phlebotomus ser- genti was the most predominant species being recorded in all localities. Abundance of Phlebotomine sand fly species, collected by different traps is shown in Table 2. More than 89.5% of specimens belong to genus Phlebotomus and 62.6% of collected spec- imens belong to subgenus Paraphlebotomus. Frequency of sand flies by genus and percent collected by different traps are shown in table 3. CLT (22.6%) and SPT (22.3%) caught sig- nificantly more sand flies and ABT (7%) and WST (7.6%) caught significantly fewer sand flies than the other traps. Statistical analysis showed significant difference between collec- tion methods and genus of collected sand flies, χ2= 577.8 and p< 0.001. The sex ratios were different in phlebotom- inae sand fly species. Sex ratios in Ph. ser- genti, Ph. alexandri and Ph. papatasi were 130, 166 and 75 respectively. In Sergentomyia ge- nus sex ratio were 21 and 181 in S.sintoni,and S. sumbarica respectively. Species diversity based on Shannon-Wie- ner index and species richness has been calcu- lated to estimate species biodiversity of Phlebotomine sand flies in different collection methods in study area and is shown in Table 5. Table 1. Frequency, gender and percent of sand flies collected by different traps in three endemic Leishmaniasis Foci, North Khorasan Province, 2016 Method location A B C female male total % female male total % female male total % ABT 59 140 199 6.5 39 68 107 4.5 122 80 202 11.3 BST 183 147 330 10.7 80 85 165 6.9 245 45 290 16.2 CLT 316 328 644 21.0 401 289 690 28.8 172 133 305 17.1 DST 330 373 703 22.9 118 220 338 14.1 101 61 162 9.1 LTP 134 137 271 8.8 253 289 542 22.7 71 67 138 7.7 SPT 257 581 838 27.3 134 272 406 17.0 226 144 370 20.7 WST 30 58 88 2.9 68 76 144 6.0 204 117 321 18.0 Total 1309 1764 3073 100.0 1093 1299 2392 100.0 1141 647 1788 100.0 ABT: Animal baited trap; BST: Black Shannon trap; CLT: Light trap baited with Carbon dioxide; DST: Dis- ney trap; LTP: CDC Light Trap; SPT: Sticky paper traps; WST: White Shannon trap. A: Bacheh-Dareh; B: Kohne-Jolgeh; C: Arg Table 2. Species abundance of sand flies collected by different traps, North Khorasan Province, 2016 Species Collection methods ABT BST CLT DST LTP SPT WST Ph.sergenti 179 421 1090 918 642 869 189 Ph. papatasi 238 298 386 179 207 226 319 Ph. alexandri 20 2 94 3 23 56 15 Ph. major 3 15 11 3 6 23 0 Ph. halepensis 5 2 5 2 5 10 1 Ph. caucasicus 0 0 1 0 3 4 0 Ph. mongolensis 3 0 1 2 1 2 0 Ph. longiductus 0 0 0 2 3 0 0 Ph. turanicus 0 0 1 1 0 1 0 Ph. ansarii 0 0 0 0 0 1 0 Se. sintoni 17 18 13 41 11 219 12 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 399–406 K Arzamani et al.: Comparative Performance of … 403 http://jad.tums.ac.ir Published Online: December 31, 2019 Se. sumbarica 22 2 22 16 15 53 5 Se. pawlowskyi 2 3 0 12 7 48 0 Se. theodori 0 0 8 11 5 43 0 Se. dreyfussi 8 21 4 7 3 20 11 Se. hodgsoni 7 1 3 2 10 17 1 Se. dentata 4 0 0 4 8 17 0 Se. clydei 0 2 0 0 2 1 0 Se. grekovi 0 0 0 0 0 4 0 Frequency 508 785 1639 1203 951 1614 553 ABT: Animal baited trap; BST: Black Shannon trap; CLT: Light trap baited with Carbon dioxide; DST: Dis- ney trap; LTP: CDC Light Trap; SPT: Sticky paper traps; WST: White Shannon trap Table 3. Frequency of sand flies by genus collected by different traps, North Khorasan Province, 2016 CM Genus Total percentage Phlebotomus percentage Sergentomyia percentage ABT 448 6.9 60 7.9 508 7.0 BST 738 11.4 47 6.2 785 10.8 CLT 1589 24.5 50 6.6 1639 22.6 DST 1110 17.1 93 12.2 1203 16.6 LTP 890 13.7 61 8.0 951 13.1 SPT 1192 18.4 422 55.4 1614 22.3 WST 524 8.1 29 3.8 553 7.6 Total 6491 100.0 762 100.0 7253 100.0 CM: Collection methods; ABT: Animal baited trap; BST: Black Shannon trap; CLT: Light trap baited with Carbon dioxide; DST: Disney trap; LTP: CDC Light Trap; SPT: Sticky paper traps; WST: White Shannon trap Table 4. The sex ratio of phlebotomine sand flies collected by different traps, North Khorasan Province, 2016 CM Phlebotomus Sergentomyia Female Male Total SR Female Male Total SR ABT 191 257 448 135 29 31 60 107 BST 467 271 738 58 41 6 47 15 CLT 856 733 1589 86 33 17 50 52 DST 497 613 1110 123 52 41 93 79 LTP 424 466 890 110 34 27 61 79 SPT 342 850 1192 249 275 147 422 53 WST 284 240 524 85 18 11 29 61 Total 3061 3430 6491 112 482 280 762 58 CM: Collection methods; ABT: Animal baited trap; BST: Black Shannon trap; CLT: Light trap baited with Carbon dioxide; DST: Disney trap; LTP: CDC Light Trap; SPT: Sticky paper traps; WST: White Shannon trap Table 5. Species diversity and species richness of Phlebotomine sand flies collected by different traps, North Khorasan Province, 2016 Collection methods ABT BST CLT DST LTP SPT WST species richness 12 11 13 15 16 18 8 Species diversity based on Shannon-Wiener index 1.39 1.05 0.99 0.88 1.09 1.60 1.01 ABT: Animal baited trap; BST: Black Shannon trap; CLT: Light trap baited with Carbon dioxide; DST: Dis- ney trap; LTP: CDC Light Trap; SPT: Sticky paper traps; WST: White Shannon trap Table 2. Continued … http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 399–406 K Arzamani et al.: Comparative Performance of … 404 http://jad.tums.ac.ir Published Online: December 31, 2019 Fig. 1. Study area of Phlebotomine sand flies collection, North Khorasan Province, Iran, 2016. A: Bacheh-Dareh, B: Kohne-Jolgeh, C: Arg Fig. 2. Different collection methods used for sampling of phlebotomine sand flies, North Khorasan Province, Iran, 2016. Above: white and black Shanon and animal baited traps in location C. Below: Light trap and light trap baited with Carbon dioxide gas in location A Discussion This is the first research conducted in north- east of Iran to evaluate different sand fly col- lection methods in three endemic foci of cuta- neous and visceral leishmaniasis in North Khora- san Province, Iran. A total of 19 species of sand flies collected and identified. Some proven or suspected vectors of CL and VL in Iran in- cluding Ph. papatasi, Ph. sergenti, Ph. caucasi- cus, Ph. alexandri and Ph. major were collect- ed in the study area. Our findings confirm that these traps differ in performance and demonstrated that CLT and SPT are the most efficient sand fly collection methods. CLT collected 24.5% of all Phleboto- mus genus, 13 species and its Shannon-Wiener index was 0.99 while SPT collected 18.4% of Phlebotomus genus, 55.4% of Sergentomyia genus, 18 species of Phlebotominae sand flies and its Shannon-Wiener index was 1.60. The result of the performance of CLT and SPT is in agreement with some studies about the productivity of these methods (10, 12) but is in contrast with the result of some studies (11, 13). It seems in arid and dry area the per- formance of SPT is more appropriate than other methods, but in moisture and mountainous area this method has low efficiency. SPT become ineffective in habitats with high relative hu- midity because of the viscosity of castorl oil and only dead specimens are collected (20). In our study the performance of SPT in arid area was more than mountainous area. Species with very low abundance may be difficult to detect using non-attractive traps such as SPT. However, we captured some of rare species only with SPT, so this method could assess the species composition and is a perfect http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2019, 13(4): 399–406 K Arzamani et al.: Comparative Performance of … 405 http://jad.tums.ac.ir Published Online: December 31, 2019 method for determination of biodiversity indi- ces of phlebotominae sand flies. Collection of high abundance of Sergentomyia genus and male specimens illustrated in this method. According to our study, CLT seems to be one of the most effective collection methods and could assess the species composition of Phlebotominae sand flies. This trap showed higher performance in capturing females and Phlebotomus specimens; therefore CLT can be used for monitoring the population of Phleboto- mus genus during surveillance. It seems in mois- ture and mountainous areas this method has high efficiency and the result is in agreement with some studies. Some researchers obtained CO2 for CLT using dry ice, which is difficult for regulation of gas emission so we used a CO2 gas tank could easily, regulated and in- creased the efficacy of this method. The performance of CLT was more than LTP. CO2 is usually a long-range attractant for sand flies, whereas light is probably perceived by sand flies at much closer range (20). Conclusion Our results suggest that CLT and SPT are suitable for entomological surveys in the coun- try. CLT is higher attractive for females and Phlebotomus genus and is an ideal method for monitoring the population of Phlebotomus ge- nus during surveillance. However, SPT is an inexpensive, convenient and easy to be used to detect the presence of sand flies at low den- sities and captured some of rare species and provide a more realistic estimation of sand flies biodiversity. Therefore SPT is probably the best choice for monitoring of Phlebotominae sand flies. 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