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

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 Province, 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 genus 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.


Introduction
Leishmaniasis is transmitted to human in 98 countries and 3 territories on 5 continents in the world. There are three main forms of leishmaniases, visceral leishmaniasis (VL), cutaneous 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 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 province (2)(3)(4)(5)(6)(7) . Phlebotominae sand flies (Diptera: Psychodidae) 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 different 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 population density of sand flies in the study area (10).
In an investigation in Morocco statistical analysis showed that significantly more number 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 methods for collection of sand flies (SPT, LTP, and CLT) were compared. CLT were more attractive for females of P. perniciosus and P. neglectus. LTP showed an intermediate efficiency 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 programs (12).
In Brazil to compare the relative attractiveness of BST and WST for sand flies, several 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 phlebotominae 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 number or show the most diversity of sand flies.

Study area
This cross-sectional study was conducted during 2016 in North Khorasan Province, between 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 28,434km 2 . The province is bordered by Turkmenistan in the North and situated in northeast of Iran. Bojnurd is the capital city of the province (Fig. 1).

Selection of study villages
Three villages in the province were selected, where human VL and/or CL had been reported in the last 5 years. Bacheh-Dareh village (A): It is located in Raz and Jargalan County, 15km from the border of Turkmenistan. Several human VL and CL cases had been report- 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. Collection site was indoors and outdoors and domestic 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. 7. Animal Baited Trap (ABT). A net trap (2×3×2 meters) was used to sample sand flies attracted 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 clockwise 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 carried 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 collection 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 accordance with the procedures approved by the Ethical Committee of North Khorasan University of Medical Sciences.

Results
A total of 7253 sand flies were collected. Frequency, gender and percent of Phlebotomine sand flies collected by different traps in three collection sites are shown in Table 1. On collection site A we collected the most abundance 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-402 http://jad.tums.ac.ir Published Online: December 31, 2019 cies collection appears in this area. Significantly fewer sand flies collected in site C with A collection of only four species and in site B eight species were caught. Phlebotomus sergenti 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 specimens 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 significantly 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 collection methods and genus of collected sand flies, χ 2 = 577.8 and p< 0.001.
The sex ratios were different in phlebotominae sand fly species. Sex ratios in Ph. sergenti, Ph. alexandri and Ph. papatasi were 130, 166 and 75 respectively. In Sergentomyia genus sex ratio were 21 and 181 in S.sintoni,and S. sumbarica respectively.
Species diversity based on Shannon-Wiener index and species richness has been calculated to estimate species biodiversity of Phlebotomine sand flies in different collection methods in study area and is shown in Table  5.

Discussion
This is the first research conducted in northeast of Iran to evaluate different sand fly collection methods in three endemic foci of cutaneous and visceral leishmaniasis in North Khorasan 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. caucasicus, Ph. alexandri and Ph. major were collected 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 Phlebotomus 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 performance 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 humidity 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 405 http://jad.tums.ac.ir Published Online: December 31, 2019 method for determination of biodiversity indices 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 Phlebotomus genus during surveillance. It seems in moisture 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 increased 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 country. CLT is higher attractive for females and Phlebotomus genus and is an ideal method for monitoring the population of Phlebotomus genus during surveillance. However, SPT is an inexpensive, convenient and easy to be used to detect the presence of sand flies at low densities 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.