Diversity and Activity Pattern of Sand Flies in Cutaneous Leishmaniasis Endemic Tribal District Khyber, Pakistan

Leishmaniasis is endemic for last more than one decade in the tribal district Khyber located in the north-west of Pakistan near Pak-Afghan border. The disease burden has increased during the time of war against terrorism in the tribal belt of Pakistan bordering Afghanistan. This study was designed to investigate various ecological and entomological aspects of sand flies in war effected district Khyber. Sand flies were collected from seven different localities of the district by flit method, CDC light traps and mouth aspirator. A total of 28 species belonging to two genera i.e., Phlebotomus represented by 12 species and Sergentomyia represented by 16 species were collected during the present study. Phlebotomus mongolensis is reported for the first time from Pakistan. Phlebotomus sergenti (48.77%) was the most abundant species followed by Phlebotomus paptasi (17.89%) , Sergentomyia baghdadis (14.53%) and Sergentomyia babu (7.22%). Shannon-Weiner Index was high due to maximum number of specimens belonging to Phlebotomus sergenti. The overall female to male sex ratio was observed to be 1:1.9. Sand fly activity was observed from April to December with population density reaching its peak in the month of August. A positive correlation was observed between sand fly abundance and climatic variables, which was significant with temperature only. The high abundance of Phlebotomus sergenti support the endemicity of anthroponotic cutaneous leishmaniasis in the study area . Presence of species of subgenera Adlerius and Larusius suggest the possibility sporadic visceral leishmaniasis in the region.


INTRODUCTION
T he Phlebotomine sand flies are distributed throughout the tropics and subtropics as well as in the temperate regions of the world. The subfamily Phlebotominae consists of nearly 1000 species classified in six genera, of which the genus Phlebotomus have 13 subgenera, Sergentomyia have ten subgenera and the genus Chinius have four species and are found in the old world. The phlebotomine sand flies are found everywhere and is a group of medically important insects responsible for the transmission of protozoan parasites and some arbo-viral infections among humans and other animals. The role of sand flies as vectors of several viral, bacterial and protozoal diseases emphasizes the significance of investigating and understanding the ecology, diversity and other biological characteristics of these insects. Many species of sand flies act as vectors of leishmaniasis. This disease is mainly present in three forms; cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL) (Jal., 2022). Zoonotic cutaneous leishmaniasis (ZCL) caused by L. major and anthroponotic cutaneous leishmaniasis (ACL) caused by L. tropica are common in Pakistan. CL is endemic in many countries of Eastern Mediterranean region (Ashford, 1996).
The sand fly fauna of Pakistan is a mixture of Palearctic, Oriental and Afro tropical regions (Lewis, 1967). The topography and climatic conditions of Pakistan provides an ideal breeding and living habitat for various species of sand flies (Lewis, 1978). Several studies have been conducted on sand flies of Pakistan, but still current literature provides insufficient information about the sand fly species diversity of this region (Qutubuddin, 1951). The work of Lewis (1967), investigating the fauna of sand flies in Pakistan and its relation to spread of leishmaniasis, has a leading role. Aslamkhan et al. (1998) reviewed literature and carried out a study to investigate the biodiversity of sand flies of Pakistan and reported 29 species in his work. Kakarsulemankhel (2003Kakarsulemankhel ( , 2004

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comprising of 37 species. Ali et al. (2016a) reported 14 species of sand flies from the newly merged Tribal district of North Waziristan. Ali et al. (2016b) identified 20 species of sand flies from districts Dir Lower and Upper and ten species of sand flies had been reported from another newly merged Tribal district, Bajaur (Wahid et al., 2020). The incidence of CL is increasing from time to time in the world, mainly due to the unlawful intrusion of people into the forests and natural habitats where the potential vectors of Leishmania and reservoir animals are present. Some environmental factors also influence the survival of these insects and they can be found only in areas where conditions are suitable in terms of temperature and relative humidity. CL has become a serious public health problem in Pakistan and is highly endemic in tribal district Khyber for last more than one decade. More than 40000 cases has been reported during 2012-2019 and the causative agent has been identified as L. tropica by using PCR (Khan et al., 2016). However, data regarding sand fly species, their relative abundance, and seasonal variation is lacking. This study aims at exploring the diversity, ecology, and seasonality of sand flies in anthroponotic CL endemic tribal district Khyber. This knowledge will help in devising an effective strategy for the control of disease in the affected area.

Historical background of the study area
Khyber Pass is located on one of the most wellknown and the most strongly secured mountain ranges on the globe (The Hindukush). It has a universal historical heritage and has made an important position throughout the known history. It is always marked mainly as a corridor, through which almost all the infiltrations to the Indian subcontinent occurred, including those by the Aryans, Alexander, Mahmud of Ghazni, Tamerlane, the Mughals and the Durrani Afghans. When NATO (North Atlantic Treaty Organization) forces under the leadership of United States, attacked Afghanistan in 2001, militants penetrated the tribal district Khyber located at the Pak-Afghan border of Pakistan. Due to the absence of an active security network in the region at that time and its rough, mountainous boundaries and territory, the area became a hotbed of the insurgents. Over 90% of the district came under control of terrorist in 2007 and the area was used by militants to launch attack on civil and military establishments of Khyber Pakhtunkhwa (KP) killing thousands of people and wounding many more. After the military operations of 2012 and 2014, Khyber has been cleared of the militants but it has destroyed the infrastructure of the area and vector borne diseases like ACL has been occurring on epidemic scale since then.

Sampling sites
Khyber district is among the most CL affected areas of Pakistan. Sampling was done from seven villages distributed in all four tehsils of the district. As most of the area of Khyber is covered with mountain ranges, the majority of the population lives along the Khyber Pass and in the plain areas touching district Peshawar and Kohat. The seven sampling sites ( for rest. These Garas are used as animal sheds and in some areas used as mixed dwellings. Gara were a rich source of sand fly collection due to microclimate they used to provide for resting sand flies.

Sampling methods
In each collection site, ten to fifteen houses were selected randomly. In each house two rooms comprising of an animal room and a human dwelling were used for the collection of adult sand flies. Each collection site was visited once in a month. Most of the rooms from where collection of sand flies was made were having mud walls and thatched ceilings. The adult sand flies were collected by using flit method, CDC light traps and mouth aspirator.

Flit method and use of aspirator
Resting sand flies from human dwelling and animal rooms were collected by using flit method (Lane and Crosskey, 1993) supplemented by mouth aspirator. Collection was conducted three to four hours after sunrise. In this method, the floor of the room was covered with black sheets (to make the sand flies easily visible with dark background) and all the insects resting inside the room were killed by making the room airtight and then spraying a fastacting volatile pyrethroid insecticide (Mortein: Reckitt Benckiser Pakistan Ltd.) for 30 sec. For this purpose, all the occupants including animals and articles like tables, chairs, exposed edibles etc., were removed from the room to be sprayed, or appropriately covered. The doors and windows were kept closed and after 10-15 min the room was opened, the sand flies from the sheets were collected with the help of fine entomological forceps and placed in a tube filled with 90% ethanol. Sometimes flitting was followed by sampling live sand flies from the room, using a mouth aspirator. The number of live specimens collected from a site were added to those collected by flitting to calculate the total number of sand flies. Collection from each room was kept in a separate tube and was labeled accordingly.

CDC light traps
The sand flies were also collected by using battery operated CDC miniature light traps (model 512 with airactuated gate system and LCS-2 photo switch; John W. Hock Company, USA). For collection, a light trap was hanged 30 inches above the ground from dusk to dawn. Sand flies captured by these traps were then transferred to 90% ethanol for further processing.

Sand fly processing
Species identification of sand flies was done based on morphological and anatomical structures which can be exposed by preparing a permanent slide of each fly. For this purpose, the head of the male was separated from the body by using fine entomological needles under a stereo microscope (SD 30, Olympus, Japan). The head was placed dorsally and body laterally, taking care that the wings and legs were properly placed. A drop of Puri's media was put on the head and body before placing the cover slip. For mounting the female, the last three abdominal segments and head were separated from the body as anatomical structures in head and spermatheca were used for identification purpose. These body parts were placed on glass slide and covered with a cover slip after putting a drop of Puri's media. The slides were kept safe for at least three weeks for clearing of the body parts. The sand flies cleared after fixing on the slides were examined individually under a binocular microscope (Nikon E-100, Japan) by using 40X objective and identified up to the species level with the help of valid taxonomic keys illustrated in Artemiev (1976) and Lewis (1982). The slides of identified specimens were catalogued, placed in slide boxes and were kept in the Vector Biology and Entomology Laboratory of Institute of Zoological Sciences, University of Peshawar as reference material.

Data arrangement and analysis
Data regarding species identification was transferred to excel sheet and was arranged according to locality and months. Following ecological indices were calculated for the overall data obtained and each collection site.
Species richness (S) = number of species in the study area.
Relative abundance (p i ) = n/N x 100 where n is the number of specimens of a particular species and N is the total number of sand flies collected from a study area. Three categories of dominance were made on the basis of relative abundance: dominant (D): >10%, subdominant (D): 1-10% and satellite (S): <1%.
Degree of presence (C) = l/L x 100 where l is the number of localities where the species was found and L is the total number of studied localities. Species found in more than 80% localities were classified as constant species (C), those captured in 60.1 to 80% localities were frequent species (F), 40.1 to 60% were classified as moderate (M) species, 20.1 to 40% as infrequent (I) species, while 0 to 20% as sporadic species (S).
Sex ratio (female/male) was calculated for each species collected during the survey.
Shannon-Weiner Index for calculating species diversity (H) = -∑p i x lnp i where ln p i is natural log of p i.
Evenness (J or E or Pie-Lou's index) (E) = H/lnS where ln S is natural log of species richness.

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Month wise abundance of sand flies was plotted against climatic variables i.e. temperature, relative humidity and rainfall using excel. Correlation coefficient and its statistical significance (P<0.05) was calculated by using online statistical software (Wessa, 2017).

Species composition of sand flies
A total of 11766 sand flies (4015 female and 7751 male) belonging to 28 species of two genera and 11 subgenera were collected as adults from the tribal district Khyber during a one-year survey. The genus Phlebotomus was represented by six subgenera and 12 species viz. six species of the sub genus Paraphlebotomus (P. alexandri, P. caucasicus, P. kazeruni, P. mongolensis, P. nuri, P. sergenti), two species of subgenus Larroussius (P. kesheshiani, P. major) and one species each of the subgenera Adlerius (P. kabulensis), Phlebotomus (P. papatasi), Euphlebotomus (specimen damaged, species identification not done) and Synphlebotomus (P. ansari). Phlebotomus mongolensis is reported for the first time from Pakistan.

Sand Fly Species Composition and Activity Pattern in Tribal District Khyber
Relative abundance, sex ratio and resting site preference Collectively the relative abundance of sand flies in the study area indicated that genus Phlebotomus (72.72%) was more abundant as compared to Sergentomyia (27.28%). Phlebotomus sergenti, P. papatasi, S. baghdadis and S. babu were the dominant species in the collection. P. alexandri, P. nuri and S. dentata were subdominant while remaining species were satellite species according to their abundance (Table I). More males were collected during the survey as compared to females with over all female to male ratio for the collection was 1:1.93. The ratio for different species ranged from 1:1 to 1:4.3 (Table  I). Slightly more sand flies (52.42%) were collected from animal rooms as compared to human dwellings (47.58%). Similar observations were reported for majority of the species (Table I). Degree of presence data showed that majority of the species were constant species, two species were frequent, four were moderate, a single species was infrequent while four species were sporadic and collected from a single collection site (Table II).

Diversity indices
Over all and site wise diversity indices including richness, relative abundance, Shannon-Weiner index, Simpson diversity index and evenness were calculated and are shown in Table III. Relative abundance value was highest for Sur Kamar (19.7%), while lowest for Shah Kass (10.1%). Shannon-Weiner index was high for all collection sites which could be explained by the presence of dominant species like P. sergenti, as this species alone represent more than 50% of the collection at each site (Table I). Due to high species richness at all collection sites, species evenness values were moderate (0.46 to 0.58) but at Qambar Khel (0.71) it was high, where species richness was lowest.

Seasonal variation in species composition and relative abundance of sand flies
Information regarding seasonal activity and relationship with different climatic variables is given in Figure 2. It has been observed that seasonal activity of sand fly extends from April to December and no sand flies were collected during the cold months of January to March, while only few specimens of sand flies were collected in the months of December and April. High population densities of sand flies were observed in monsoon and post-monsoon months from July to September. Highest densities of sand flies were recorded in the months of August and September (Table IV). Month-wise diversity indices (Table IV) also shows seasonality in occurrence of sand flies. The population of P. sergenti peaked in the month of September while that of P. papatasi peaked in the month of August. Both these important vector species occur throughout the active season of the study period. Overall, highest density of sand flies was recorded in the month of August. Very little number of specimens belonging to few species were collected in April and in the cold month of December (Table IV). Comparing sand fly abundance with climatic variables have shown that an increase in abundance was observed after rainfall during the hot months when high relative humidity coupled with favourable temperature. Relationship of abundance with climatic variables have shown a very weak and non-significant correlation with humidity and rainfall but a strong positive and significant correlation was observed for temperature (P<0.05).

DISCUSSION
The ecology of Pakistan supports a variety of insect species including sand flies and mosquitoes and has been endemic for vector borne disease like malaria, dengue, chikungunya and leishmaniasis (Zaidi et al., 2017). According to provincial health department of Khyber Pakhtunkhwa more than 40,000 cases of CL have been reported from tribal district Khyber alone during the past O n l i n e

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Sand Fly Species Composition and Activity Pattern in Tribal District Khyber 7 one decade. The present study is the first detailed research work on the ecology of sand flies in different biotopes of rural communities of district Khyber; a major focus of ACL in the northwest of Pakistan. Our study yielded a total of 28 species of sand flies. Eleven species belonging to the genus Phlebotomus and sixteen to Sergentomyia were collected from indoor-resting sites in seven selected localities of tribal district Khyber. Phlebotomus. mongolensis is reported for the first time from Pakistan, thus adding one more species to the sand fly checklist of Pakistan and increasing the number of species in genus Phlebotomus to 26 and overall species number to 51. Species reported in current study had been reported in other studies conducted in the hilly areas of northern Pakistan (Munir, 1994), surveys conducted by Aslamkhan (1998), from Balochistan (Kakarsulemankhel, 2003(Kakarsulemankhel, , 2004, Dir (Ali et al., 2016b), North Waziristan (Ali et al., 2016a), Bajaur (Wahid et al., 2020), Chitral and Nowshera (Rasheed et al., 2023). Phlobotomus. mongolensis occurrence in Pakistan was predicted by Lewis (1967), but it is now reported for the first time in current study from endemic area of district Khyber. This species inhabits highlands with semi desert like conditions as found in the current study. Sergentomyia mervynae, S. murgabiensis and S. indica are reported for the first time from the province of Khyber Pakhtunkhwa.
Relative abundance data has shown that sand flies of genus Phlebtotomus (72.72%) were more abundant as compared to Sergentomyia (27.28%) in the collection done from tribal district Khyber of Pakistan. Similar results have been reported in other studies carried out in leishmaniasis endemic areas of Pakistan (Ali et al., 2016a, b;Wahid et al., 2020). Among the 27 species reported in present study Phlebotomus sergenti (48.77%) was the most abundant species. Overall value of Shannon-Weiner index as well as for each site has been pointing towards the presence of a single species constituting a large proportion of the sand fly population (Al-Koleeby et al., 2022) like P. sergenti in present study. Studies conducted in Pakistan have also reported similar results. Similar pattern of relative abundance has been reported from the tribal districts of North Waziristan (Ali et al., 2016a) and Bajaur in Pakistan (Wahid et al., 2020). Contrary to our findings, a study conducted in district Dir reported P. salengensis, a mountain dwelling species highest in number followed by P. sergenti, while P. papatasi was among the least occurring species in the district Dir (Ali et al., 2016b). This could explain the endemicity of urban cutaneous leishmaniasis in district Khyber and North Waziristan and sporadicity of the disease in Dir (Rowland et al., 1999) where in the later, the vector species (P. sergenti and P. papatasi) were present in low abundance.
In contrast to the current work, the study conducted in Iran (Hazratian et al., 2011;Kassiri and Javedian, 2012;Vatandoost et al., 2018;Vahabi et al., 2016), Turkey (Kasap et al., 2009) and Jordon (Kamhawi et al., 1995) have reported P. papatasi as the most abundant species as compared to P. sergenti. Phlebotomus papatasi had been incriminated as proven vector of L. major (Maroli et al., 2013;Vahabi et al., 2016) and in all these studies where P. papatasi has been reported to be more abundant were conducted in rural cutaneous leishmaniasis endemic area where L. major was the disease-causing agent (El-Sawaf et al., 1989;Yaghoobi-Ershadi and Javedian, 1995;Vahabi et al., 2016). As current study area is endemic for L. tropica (Khan et al., 2016), which could explain the abundance of P. sergenti as compared to P. papatasi, secondly P. sergenti is a mountainous species while P. papatasi occur in plain areas, which could also explain the abundance of P. sergenti or P. papatasi on the basis of topography in the above-mentioned studies (Kasap et al., 2009;Kassiri and Javedian, 2012). Among the seven collection sites of the present study no major differences in diversity indices have been observed among these sites. Species richness was observed to be same in all collection sites except Qamber Khel. Qambar Khel is a plain area while remaining sites are foot hills which resulted in differences in the diversity and abundance of sand flies at this collection site. Differences were also present in abundance of sand flies in Qambar Khel and other areas. Phlebotomus papatasi is a species of plain area (Kassiri and Javedian, 2012;Vatandoost et al., 2018), which explains its abundance in Qambar Khel, while species like P. sergenti, P. alexandri, P. mongolensis, P. major, S. sumbarica and other species that prefer hilly areas were absent or reported in less abundance from Qambar Khel (Kassiri and Javedian, 2012). Qambar Khel was also having a more urban set up with less animal sheds for keeping cattle, goat and sheep which along with urbanization, topography and habitat scarcity could explain low diversity of sand flies at this site. Remaining six sites were having almost the same habitat and differences in diversity of species exist only for those that were less abundant in the collection (Table I).
Two types of indoor resting sites i.e., animal rooms and human dwellings were used for collection of sand flies. More sand flies were collected from animal rooms (52.42%) than human dwellings (47.58%). Though this difference was small, but could be attributed to less disturbance, more resting sites and construction type of animal rooms (mostly mud walls and thatched roofs), that can provide better resting sites to these flies during daytime. Other reasons may include increase in human population which has changed host preferences from wild animals to human and domesticated animals and shift in breeding places from rodent's burrows and wild animal O n l i n e

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resting sites to livestock ranches, barns and animal rooms in domestic dwellings. Presence of cattle and domestic animals in previously uninhabited areas not only replace rodents and wild animals as food but has also shifted breeding places of sand flies to cattle sheds (Vatandoost et al., 2018). Sex ratio of sand flies tends to remain 1:1 in nature (Killick-Kendrick, 1999) but our study has reported more males as compared to females. The overall female to male ratio was 1:1.9 for the study period in district Khyber. Important vector species like P. sergenti and P. papatasi have sex ratio of 1: 2.4 and 1: 2.2, respectively. Similar observations had been reported by other studies conducted in Pakistan where more males were collected as compared to females (Ali et al., 2016a, b;Wahid et al., 2020). Presence of higher frequency of males as compared to females could be attributed to various factors like presence of indoor larval habitats (Vatandoost et al., 2018), availability of more host for blood feeding to females, which attract males also as males arrive first at the feeding site seeking for a female to mate with (Dye et al., 1991). Studies had shown that at higher densities of sand flies the sex ratio is more male biased (Quinnell and Dye, 1994). In our study area, people keep their animals indoor during the daytime to protect them from the heat of hot summers and outdoor at night. As current study used flit method for collection of sand flies from their indoor resting sites in human dwellings and animal rooms after sun rise and before sunset, female were taking shelters in these rooms followed by males which could be the probable reason for male biased sex ratio (Kasap et al., 2009).
Knowledge regarding distribution, abundance and seasonal variation help in deciding the correct time and strategy to implement control programs in an area (Galvez et al., 2010). Present study shows a single peak of abundance during the month of September and occurrence only during the warm months and relatively cold months of November and December. Seasonal activity of sand flies is greatly influenced by climatic variables like temperature, relative humidity and rainfall (Maroli et al., 2013). In district Khyber there are two spells of rain, one is winter and the other is summer. Winter spell occur in the month of January and summer during the monsoon period from July to September, however, a positive effect of temperature along with relative humidity on sand fly population has been observed during the monsoon rains of summer. A positive correlation of temperature with population abundance has been reported for sand flies (Gebresilassie et al., 2015). An increase in abundance of overall sand flies has been observed during these months with population density reaching its maximum in August, when temperature and relative humidity both were comparatively high as compared to other months. Similar results have been reported in other parts of the country where single peak of sand flies was observed during the active season. In North Waziristan the active season has been reported to be shorter than present study from April till September with a peak in the month of July (Ali et al., 2016a) and in Dir also the active season ranged from May to October with a peak in July and August (Ali et al., 2016b). In district Bajaur unimodal distribution with active season ranging from June to October has been reported by Wahid et al. (2020). Temperature and specifically relative humidity had been observed to play an important role in the seasonal activity of sand flies in such areas (Srinivasan et al., 2013).
It has been reported that in colder climatic zones sand flies have shorter active season (Hazratian et al., 2011;Ali et al., 2016a, b), while area with warm temperate climate have lengthy active season (Mirhoseni et al., 2017). Studies have shown that in arid and semi-arid biotopes sand flies have short active season with bimodal distribution while warm temperate areas have lengthy active season with unimodal distribution (Hazratian et al., 2011). District Khyber lies in temperate warm zone, which explains lengthy active season with single density peak immediately after rainy season due to high relative humidity coupled with high temperature resulting in increase in abundance of sand flies during these months.

CONCLUSION
Among the reported species P. papatasi, P. sergenti, P. alexandri, P. salehi, P. keshishiani and P. major (Kassiri and Javedian, 2000) had been proven as vectors of various Leishmania parasites in other parts of the world. This shows that our study area is at risk of an increase in disease incidence and spread of Leishmania major and visceral leishmaniasis. In conclusion, the abundance of P. sergenti as compared to other sand fly species such as P. patasi, the geographical conditions and circulation of L. tropica in the local population explain the endemicity of ACL or urban cutaneous leishmaniasis in district Khyber. The low abundance of sand fly species of subgenera Larusius and Adlerius in this geographical region shows the possibility of sporadic VL cases in the region. More epidemiological studies are required along with continuous vector surveillance in the field of cutaneous as well as visceral leishmaniasis to decrease the disease incidence of CL and prevent cases of VL in the area.

Funding
The funds were provided by Higher Education Commission of Pakistan for funding this study under NRPU project No. 4135.

IRB approval and ethical approval
This study was reviewed, approved and monitored by the Ethical Committee, Faculty of Life and Environmental Sciences, University of Peshawar, under applicant certificate number (12/EC/F.LIFE-2020).

Statement of conflict of interest
The authors have declared no conflict of interest.