J Arthropod-Borne Dis, June 2022, 16(2): 159–172 N Hosseini-Vasoukolaei et al.: Bioecological Study on … 159 http://jad.tums.ac.ir Published Online: June 30, 2022 Original Article Bioecological Study on the Sand Flies (Diptera: Psychodidae, Phlebotominae) in Sari County, North of Iran *Nasibeh Hosseini-Vasoukolaei1, Leila Ghavibazou2, Amir Ahmad Akhavan3, Ahmad Ali Enayati1, Elham Jahanifard4, Mahmoud Fazeli-Dinan1, Jamshid Yazdani-Charati5, Seyed Hasan Nikookar1, Zahra Saeidi3, Atieh Shemshadian1 1Department of Medical Entomology and Vector Control, Health Science Research Centre, Faculty of Health, Mazandaran University of Medical Sciences, Sari, Iran 2Research Committee, Faculty of Health, Mazandaran University of Medical Sciences, Sari, Iran 3Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 4Department of Medical Entomology and Vector Control, School of Public Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 5Department of Biostatistics, Health Sciences Research Center, Addiction Institute, Mazandaran University of Medical Sciences, Sari, Iran *Corresponding author: Dr Nasibeh Hosseini-Vasoukolaei, Email: nasibeh.hoseini@gmail.com (Received 25 Nov 2020; accepted 11 June 2022) Abstract Background: Phlebotomine sand flies are vectors of Leishmania species, the causative agents of leishmaniasis in the world. Present study aimed to evaluate the bioecological aspects of sand flies in different ecotopes in Sari County, north of Iran. Methods: Sand flies were collected from four villages in mountainous, forest, plain and peri-urban areas monthly using sticky traps in May–October 2016. Mounted specimens were identified using valid identification keys under optical microscope. The Arc GIS 10.5 software was applied for showing the distribution of sand flies. Shannon-Weiner, Simp- son and Evenness species diversity indices were calculated. Results: Generally, 334 specimens were captured and identified, namelly Phlebotomus kandelakii, Ph. papatasi, Ph. major, Ph. sergenti, Ph. longiductus, Ph. halepensis, Ph. tobbi, Sergentomyia dentata, Se. theodori, Se. sintoni, Se. an- tennata and Se. sumbarica. The most common species was Ph. kandelakii (n= 128, 38.32 %). The highest Simpson in- dex (0.81) and abundance (N= 141) were recorded in the mountaineous area. Shannon diversity index was higher in the forest (H'= 1.53) and the highest evenness index was in the plain area (J'= 0.93). The highest richness (S= 9) and Shan- non indices (H'= 1.57) were observed in June. Conclusions: Phlebotomus kandelakii, Ph. sergenti, Ph. tobbi, Ph. longiductus, Se. theodori, Se. antennata and Se. sumbarica were recorded for the first time in the study area. Since some species are incriminated for leishmaniasis transmission, further studies are required in the northern regions of Iran to timely control measures planning. Keywords: Sand fly; Ecology; Biodiversity; Northern Iran Introduction Sand flies belong to the order Diptera, sub- order Nematocera, family Psycodidae and sub- family Phlebotominae (1, 2). They are vectors of various types of leishmaniasis. It is a ne- glected tropical disease caused by obligate pro- tozoan parasites of the genus Leishmania (Tryp- anosomatida: Trypanosomatidae) transmitted by the bites of infected female sand flies that host animals such as canids, rodents, marsupials, hy- raxes or humans (2, 3). Leishmaniasis is endem- ically extended to extensive areas of the trop- ics, subtropics and Mediterranean basins of about Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:nasibeh.hoseini@gmail.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2022, 16(2): 159–172 N Hosseini-Vasoukolaei et al.: Bioecological Study on … 160 http://jad.tums.ac.ir Published Online: June 30, 2022 102 countries, with a total of 350 million peo- ple at risk and 12 million infected (2, 4, 5). Al- most 53 species of Leishmania have been pre- sented in diferrent parts of the world. Of these, 31 species are mammalian parasites, and 20 species are pathogenic to humans. Leishmania parasites cause four main clinical types of vis- ceral, cutaneous, diffuse cutaneous and muco- cutaneous leishmaniasis (6). Cutaneous leish- maniasis is the well-known form of the dis- ease and is estimated to occur 87% of its glob- al incidence in ten countries including Afghani- stan, Algeria, Colombia, Brazil, Iran, Syria, Lib- ya, Tunisia, Pakistan and Iraq (7). Phlebotomine sand flies are belonging to two genera of Phlebotomus in the old world and Lutzomyia in the new world. According to the latest classification, 53 species of sand flies have been documented from different parts of Iran, including 34 Phlebotomus species of six subgenera and 19 Sergentomyia species of six subgenera (8). Phlebotomus sergenti ser- genti and Ph. sergenti similis were considered as subspecies, and the presence of Ph. (Adler- ius) kabulensis, Ph. (Adl.) salangensis, Ph. (Adl.) turanicus, Ph. (Larroussius) langeroni, Ph. (Transphlebotomus) mascittii, Ph. (Lar.) smirnovi, Ph. (Euphlebotomus) caudatus and Se. (Grassomyia) indica were doubtful in the country (9). Phlebotomus (Adl.) comatus is re- ported as the new species from northwest of the country by Zahraei-Ramazani et al. (10). Sand flies are commonly observed and adapted to natural environments (human and animal places), especialy Ph. papatasi and Ph. sergenti. This may be advantageous due to the potential breeding and resting sites and easy food source, which can ultimately enhance the risk of transmitting pathogens to humans and domestic animals (11). Also, various environ- mental and physiological conditions of sand flies can influence their capacity to transmit the parasite (12). Therefore, understanding the biological and ecological aspects of sand flies is essential. Geographic information systems (GIS) are software tools regarded to determine the prob- ability of presence, biodiversity, distribution and abundance of vectors and to predict the risk map of vector-borne diseases (13, 14). Spatio- temporal epidemiological researches by GIS can be applied to predict disease distribution in the regions at risk (15). GIS technique is also ap- plied for insect ecology by combining climatic and insect parameters to assess pest risk areas (16). Biodiversity is the set of differences be- tween organisms at all levels of the life spec- trum, from genes and species to higher levels of taxonomy, including habitat and ecosystem types, and is represented in different types of Alpha (α), Beta (β) and Gamma (Ϫ). It is an important ecological issue because changes in the biodiversity of communities can propably lead to the emergence of some dominant species, create new ecological niches for proliferation of them, and enhance the prevalence of sand fly-borne diseases (17). No studies have been conducted on the biodiversity of sand flies in Mazandaran Province. Therefore, the study of biodiversity in the region is momentous and can clarify the transmission of disease to humans and reservoirs. Mazandaran Province is considered as one of the sporadic foci of leishmaniasis. In pre- vious studies, sporadic cases of cutaneous and visceral leishmaniasis have been documented in some parts of the Province (18-20), some of which have a history of travel to endemic areas of leishmaniasis. Since Mazandaran Prov- ince borders with the provinces that are en- demic foci of the disease, disregarding the char- acteristics of the vectors can lead to an increase in the incidence of this disease. Few studies have been carried out in the area to identify vec- tors that complete the disease cycle. Therefore, this study focuses on collecting sand flies in Sari County of Mazandaran Province and iden- tifying their ecological and biological char- acteristics. This data set will serve as the basis for other epidemiological studies and the de- sign of disease control strategies. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 159–172 N Hosseini-Vasoukolaei et al.: Bioecological Study on … 161 http://jad.tums.ac.ir Published Online: June 30, 2022 Materials and Methods Study area This descriptive cross-sectional study was conducted from May to October 2016 in Sari County, the capital of Mazandaran Province, which is located in the central part of the province, on the foothills of Alborz Mountains in north of Iran with a longitude of 53°5 and a latitude of 36°4. The county is divided into two major mountainous and plain areas. Sari has a moderate and humid climate in summer and a relatively cold climate in winter. The southern mountainous part of the county has rather long and very cold winters. The aver- age daily temperature and annual rainfall were reported 17.7 °C and 824.4mm, respectively. The maximum and minimum temperatures were in August (36.6 °C) and December and Feb- ruary (2.7 °C), respectively. The maximum and minimum average daily relative humidity were 82% in November and 72% in September, re- spectively. In this study, sampling areas were selected in such a way that appropriate coverage in the entire study area is ensured by considering the diversity in terms of geography and type of eco- system. Accordingly, four villages were selected from each mountainous (Era), forest (Shekta), plain (Kordkheil) and, peri urban (Zoghal Chal) areas of Sari County (Fig. 1). In each area, al- titude, longitude and latitude were recorded using a global positioning system (GPS) device. In addition, due to the lack of information about the distribution of leishmaniasis vectors in Sari, the areas in which the health center had reported the admission of native leishmaniasis patients in the past years, received more attention. Sand fly collection (Sampling) and identifi- cation In this study, sampling was conducted once a month to determine the seasonal and month- ly activity of sand flies from the beginning to the end of the sand fly activity season. Speci- mens were collected using sticky paper traps coated with castor oil. During sampling in each selected area, three permanent stations were selected for trapping and 60 sets of sticky traps were installed per station: 15 traps in human in- door places (bathrooms, bedrooms, warehouses), 15 in animal indoor places (stables, poultry hous- es, animal habitats) and 30 in outdoors (yards) before sunset and picked up the next morning before sunrise. Sand fly specimens were taken from sticky paper using a fine needle, washed in acetone, and stored in small vials with 70% ethyl alcohol, then transferred to the Depart- ment of Entomology, Faculty of Health, Ma- zandaran University of Medical Sciences for determination of abdominal condition, mount- ing and identification. The main morphological characters used in this study were pharyngeal ar- mature, cibarial teeth, female spermatheca and male genitalia. To determine the physiological condition of the abdomen, each sample was dis- sected separately in physiological serum using dissecting needles under a stereomicroscope. To prepare a permanent microscopic slide, the head and the last three segments of the abdo- men were seperated. The isolated pieces were then mounted on a clean slide in Puri me- dium. In the case of the genus Phlebotomus, the head was mounted upwards and in the case of the genus Sergentomyia, the head was mounted downwards on the slide (21). The mounted slides were immediately transferred to an incubator at 37 °C. After drying the slides were identified according to valid mor- phological keys (22). GPS points of the collected sand flies were used as a layer for analysis. Arc GIS 10.5 soft- ware was used to prepare the distribution map of sand flies in the study area. The GIS layer containing sand fly data was transferred to Arc Map which is the main component of Arc GIS software for geospatial processing. Statistical analyzes were performed using SPSS v.20 (IBM, New York, USA). Nonpara- metric Chi-Square and Binomial statistical tests were used to compare the data. P values less than 0.05 were considered significant. http://jad.tums.ac.ir/ https://en.wikipedia.org/wiki/Alborz_Mountains J Arthropod-Borne Dis, June 2022, 16(2): 159–172 N Hosseini-Vasoukolaei et al.: Bioecological Study on … 162 http://jad.tums.ac.ir Published Online: June 30, 2022 Biodiversity of species and statistical analyses The number of taxa (S), number of individ- uals (n), Simpson’s diversity index (D), Shannon’s diversity index (H) and Pielou’s evenness index (J) were calculated to estimate Alpha-biodiversity with the following equa- tions: Shannon index: Where, pi is the relative abundance of the ith species (n/N), N; the total number of col- lected individuals. Evenness index (Pielou’s index formula): Where, H' is the Shannon-Wiener function and S is the total number of species observed. Simpson’s index: where, Pi=ni/N; ni, the relative abundance of a species calculated as the proportion of in- dividuals of a given species to the total number of individuals in a community, N (23). Software past- v.3 (Paleontological Statistics Software Package) was applied to perform whole developed calculations (24). Results A total of 334 sand flies were collected using sticky traps from May to October 2016 in the study area. Two hundred and four (61. 07%) specimens belonged to genus Phleboto- mus and 128 (38.32%) specimens to genus Ser- gentomyia, including six subgenera of which 146 (43.71%) subgenus Larroussius, 123 (36. 82%) Sergentomyia, 52 (15.56%) Phlebotomus, 2 (0.52%) Adlerius, 1 (0.29%) Paraphleboto- mus, and 1 (0.29%) Parrotomyia (nine samples (2.69%) were undetectable) (Table 1). In the present study, among 12 species of Phlebotominae, the most frequent Phlebotomus species was Ph. kandelakii (38.32%) followed by Ph. papatasi (15.56%) and Ph. major (5.08 %). Sergentomyia dentate with 22.45% (n= 75) was the most frequent Sergentomyia sand flies followed by Se. theodori (n= 27, 8.08%) and Se. sintoni (n= 15, 4.49%) (Table 1). In terms of gender, out of 334 phlebotom- inae, 194 (58.08%) were identified male and 140 (41.91%) were female. The male to female ratio was 1:0.72. This difference was statisti- cally significant (P= 0.004). From the perspective of abdomen physio- logical condition, among the 140 female sand flies, 18 (12.85%) were fed, 120 (85.71%) were unfed, and 1 (0.71%) was semi-gravid and the abdomen physiological condition was unclear in one sample because of color change (Table 1). This difference was significant (P= 0.004). The results showed that 107 (46.3%) sand flies were collected from human hosts and 124 (53. 7%) from animal hosts. In total, 231 (69%) sand flies were collected from indoors and 103 (31%) were from outdoors (P= 0.004). The most prevalent species were Ph. kandelakii and Se. dentata with rate of 34.19% and 23.8% in indoors, and 47.57% and 19.41% in outdoors, respectively (Table 1). Genus Phlebotomus was mostly observed in indoors (141/204, 69.1%) (P= 0.004). Phlebotomus kandelakii, Ph. pa- patasi, and Ph. major were caught more in in- door places, and consequently, they have more access to human and animal hosts (Table 1). The activity of sand flies started in early- May and ended in late-October. The highest abundance of sand flies was observed in June (n= 102, 30.53%) and July (n= 155, 46.40%) (P= 0.004). Among the collected species, Ph. papatasi was active in all months of the year. The highest prevalence of Ph. kandelakii was observed in July (n= 124, 80%) and Ph. papa- tasi and Se. dentata in June (Fig. 2). Analysis of alpha-biodiversity indices showed that the highest richness (S) was ob- served in Shekta area (S= 8) and in June (S= 9), while the lowest was found in Kordkheil (S= 3) and in October (S= 2). The highest value of Shannon was in Shekta (H'= 1.53) and even- ness was in Kordkheil (J'= 0. 93) and both in May (H'= 1.57, J'= 0.95). The maximun val- ues of Shannon were presented in Shekta and http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 159–172 N Hosseini-Vasoukolaei et al.: Bioecological Study on … 163 http://jad.tums.ac.ir Published Online: June 30, 2022 May, whereas Simpson index indicated that the highest dominance was in Era (D= 0.81) and in July (D= 0.67), due to the presence of the dominant species Ph. kandelakii. Other data on the status of sand fly biodiversity by region and month are shown in Table 2 and 3. Figure 3 shows that Ph. kandelakii, Ph. papatasi and Se. dentata are the most preva- lent species in Era, Zoghal Chal, Shekta and Kordkheil areas, respectively. Phlebotomus lon- giductus, Ph. halepensis, and Se. sumbarica were collected only from the mountainous area (Era) and the foothills. While Ph. kandelakii and Ph. major were collected from both mountainous area (Era) and forest (Shekta). Phlebotomus pa- patasi and Se. antennata were collected from both forest and peri urban areas (Zoghal Chal). Sergentomyia theodori, Se. dentata, and Se. sin- toni were collected from three areas: the plain (Kordkheil), forest and peri urban. Phlebotomus sergenti was collected from forest and Ph. tobbi from peri urban area. Temporal distribution of Ph. kandelakii in the study region is shown in Figure 4. This species was not collected from study area in May. Table 1. Phlebotomine sand flies according to species, sex, and collection places in Sari County, north of Iran, 2016 Species Human places Animal places Outside places Total Male Female Male Female Male Female N % Phlebotomus (Larroussius) kandelakii 15 19 21 24 36 13 128 38.3 Sergentomyia (Sergentomyia) dentata 7 22 13 13 7 13 75 22.5 Phlebotomus (Phlebotomus) papatasi 8 3 30 3 5 3 52 15.6 Sergentomyia (Sergentomyia) theodori 6 4 4 1 10 22 27 8.1 Phlebotomus (Larroussius) major 5 4 2 3 3 0 17 5.1 Sergentomyia (Sergentomyia) sintoni 5 3 2 0 4 1 15 4.5 Sergentomyia (Sergentomyia) antennata 2 1 2 0 1 0 6 1.7 Phlebotomus (Adlerius) longiductus 0 0 0 0 1 0 1 0.3 Phlebotomus (Adlerius) halepensis 0 0 1 0 0 0 1 0.3 Phlebotomus (Larroussius) tobii 0 0 1 0 0 0 1 0.3 Phlebotomus (Paraphlebotomus) sergenti 0 0 0 0 0 1 1 0.3 Sergentomyia (Parrotomyia) sumbarica 0 0 0 0 0 1 1 0.3 Unknown 0 3 2 2 1 1 9 2.7 Total 48 59 78 46 68 35 334 100.0 Fig. 1. Sampling sites in Sari County, north of Iran http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 159–172 N Hosseini-Vasoukolaei et al.: Bioecological Study on … 164 http://jad.tums.ac.ir Published Online: June 30, 2022 Fig. 2. Monthly frequency and percentage of sand flies, collected in Sari County, north of Iran, 2016 Fig. 3. Geographical distribution and frequency of phlebotomine sand flies in Sari County, north of Iran, 2016 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 159–172 N Hosseini-Vasoukolaei et al.: Bioecological Study on … 165 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 2. Biodiversity indices of sand flies, based on spatial distribution in Sari County, north of Iran, 2016 Zoghal Chal Kordkheil Shekta Era Biodiversity indices 6 3 8 5 Number of species (S) 45 10 129 141 Abundance (N*) 1.27 1.03 1.53 0.39 Shannon (H') 0.35 0.38 0.28 0.81 Simpson (1-D) 0.59 0.93 0.59 0.29 Evenness (J') *: Nine unknown specimens were not included in the biodiversity index analysis Fig. 4. Temporal distribution of Phlebotomus kandelakii in Sari County, north of Iran, a-f: May-October 2016 [a] [b] [c] [d] [e] [f] http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 159–172 N Hosseini-Vasoukolaei et al.: Bioecological Study on … 166 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 3. Biodiversity indices of sand flies based on temporal distribution in Sari County, north of Iran, 2016 October September August July June May Biodiversity indices 2 5 7 7 9 5 Number of species (S) 2 17 50 153 97 6 Abundance (N*) 0.69 1.1 1.4 0.75 1.57 1.57 Shannon (H') 0.5 0.45 0.34 0.67 0.27 0.22 Simpson (1-D) 1 0.61 0.45 0.30 0.54 0.95 Evenness (J') *: Nine unknown specimens were not included in the biodiversity index analysis Discussion The study of leishmaniasis vectors is nec- essary as a prerequisit to many epidemiologi- cal studies. The results of this study can in- form authorities to make evidence-based deci- sions regarding appropriate interventions to control vectors and the diseases they transmit. In the present study, seven Phlebotomus species and five Sergentomyia species were col- lected. This is the first report after 1998 (25) on different biological and ecological aspects of sand flies in Sari County, Mazandaran Prov- ince, north of Iran. In the study of Rahbarian et al. in Mazanda- ran Province, a total number of 194 sand flies were collected which included six species of Ph. major, Se. sintoni, Ph. halepensis, Ph. an- drejevi, Ph. papatasi, and Se. dentata (25). Com- pared to previous studies, in the present in- vestigation Ph. kandelakii, Ph. sergenti, Ph. tobbi, Ph. longiductus, Se. antennata, Se. the- odori, and Se. sumbarica were reported as new records in Mazandaran Province. Other related studies in northern Iran were recently conducted in Gilan Province, neighboring Ma- zandaran Province, in which eleven species of sand flies were reported (26, 27). In the present study, twelve species of sand flies were identi- fied, so the species richness is higher than the previous studies, it probably indicates a thor- ough sampling effort in the present study. Regarding the sex of collected sand flies in this study, the sex ratio (male: female) was 1:0.7, which indicates that male sand flies were collected 1.4 times more than female sand flies. In accordance with this study, previous studies reported that the male to female ratio was 1:0.74 in Thiland (28), 1:0.92 in western Saudi Ara- bia (29) and 1:0.5 in central Iran (30). Another study in norhtwest of Iran reported a higeher sex ratio than the current study, in which male sand flies were captured 2.5 times more than female sand flies (31). The sticky paper trap method used in our study showed that males were more attracted to traps than females. An- other study in Thiland using the method of CDC light trap, suggested that the frequency of collected male sand flies was higher than that offemales, which could be explained by the natural behavior of males that follow females to mate (28). In terms of the abdomen physi- ological condition, in our study most of the captured female sand flies were unfed, showing that they were more active and flew more than fed ones, probably for sugar or blood feeding. One of the main findings of this study was the abundance of species such as Ph. kandelakii, Ph. papatasi, and Ph. major that are either prov- en or possible vectors of leishmaniasis in the world (32, 33). Additionally, Se. sintoni and Se. dentata were reported as lizard Leishmaniasis (34). According to the current study, Ph. kan- delakii was the most abundant species collect- ed in indoors and outdoors in the study area. The value of Simpson dominance Index was high in some areas of study because of the abundance of this species. The higher domi- nance value in a region may indicate the high- er potential of vector for disease transmission. The first report of visceral leishmaniasis in http://jad.tums.ac.ir/ file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_25 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_25 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_26 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_27 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_28 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_29 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_30 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_31 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_28 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_32 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_33 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_34 J Arthropod-Borne Dis, June 2022, 16(2): 159–172 N Hosseini-Vasoukolaei et al.: Bioecological Study on … 167 http://jad.tums.ac.ir Published Online: June 30, 2022 Iran was reported from Mazandaran Province in northern Iran in 1949 (35). Until recently, visceral leishmaniasis was reported only based on epidemiological evidence and the high prev- alence of sand fly vectors of Kala Azar. In pre- vious studies, Leishmania promastigote infec- tion was identified in Ph. kandelakii in the en- demic focus of Meshkin-Shahr in northwest- ern Iran (36). In latter studies, L. infantum was detected in Ph. kandelakii using molecular meth- ods at the same focus in northwestern (37) and in northeastern Iran in North Khorasan Prov- ince (38). Thus, according to previous studies, Ph. kandelakii, the most abundant species collected in this study area, is of paramount importance considering the fact that it is a vector of ZVL in Iran. Phlebotomus papatasi was another species collected in this study. Various studies have shown that Ph. papatasi is the main and prov- en vector of ZCL which is distributed all over the country (9, 32). In the past studies, the parasitic infection of Ph. papatasi by L. ma- jor, L. turanica and L. gerbilli were identified (39). Presence of non-pathogenic L. turanica in Ph. papatasi and animal reservoirs can im- ply the role of this parasite in sustaining the disease cycle (40-44). Another species collected in the current study was Ph. major, which was reported from 17 out of 31 provinces of Iran with human cases of ZVL and according to the current study, this species was mostly collected from mountainous areas (32, 45). Previous morpho- logical and morphometric study in the north- west of the country, showed other morphotypes including Ph. major neglectus and Ph. major krimensis for this species (46). It is not usual- ly difficult to identify the male Laroussius sand flies, but females of some species are not eas- ily distinguishable. Further studies using mor- phological and molecular data are suggested. In the present study, Ph. kandelakii, Ph. papatasi and Ph. major were mainly collected from indoor places, thus they had more access to human and animal hosts. In accordance with the current study, in an investigation on Phlebotominae ecology in an endemic cutane- ous leishmaniasis focus in Isfahan conducted by Yaghoobi Ershadi in 2001, the most preva- lent specie in indoors was Ph. papatasi (47). In another study in south of Iran, Ph. papatasi was collected mostly from indoors (48). In the studies conducted in Qom Province (49), cen- tral Iran and in Ardabil Province (31), north- west of the country, Ph. kandelakii was collect- ed more from indoors.This finding could indi- cate the habituation of these species to human habitats. In this study, Ph. kandelakii was collected from the mountains and peaked in June, while Ph. papatasi was collected from areas other than mountains. According to previous stud- ies, Ph. papatasi usually prefers the plains over the mountains (50, 51). In the present study, the highest temporal distribution was associated to Ph. papatasi, which was collect- ed in all months of sand fly activity in the area. In the current study, sand fly activity began in May and continued until October, while the peak of seasonal activity was in July. Accord- ing to our previous study in the same study ar- ea, in November, more disease transmission and higher incidence rate of leishmaniasis oc- cured after the incubation period, from sand fly bites to nodule development (19). During a study conducted in an endemic cutaneous leishman- iasis focus in Golestan province, north east of Iran, sand flies were collected in 7 months of the year and the activity of sand flies was identi- fied from May to November (52). It has been admitted that phlebotomine sand flies have a more or less definite sea- sonal distribution pattern in Mediterranean countries, become visible in late spring or early summer, and are active until early autumn (53, 54). However, various studies have verified the bimodal pattern of temporal dispersion of phlebotomine sand flies with one peak in spring and another in summer or autumn (11, 55, 56). One of the main findings of this study was the collection of eight species of Phlebotom- http://jad.tums.ac.ir/ file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_36 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_37 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_38 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_9 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_32 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_39 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_40 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_32 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_45 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_46 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_47 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_48 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_49 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_31 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_50 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_51 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_19 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_52 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_53 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_54 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_11 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_55 file:///D:/ /jad2/NO.%2044/1337-1371%20%20%20hosseini-vasoukolaei/author%20%20%2001.10.19/Dr%20Hosseini-Vasoukolaei.doc%23_ENREF_56 J Arthropod-Borne Dis, June 2022, 16(2): 159–172 N Hosseini-Vasoukolaei et al.: Bioecological Study on … 168 http://jad.tums.ac.ir Published Online: June 30, 2022 inae from the forest, five species from the mountains, three species from the plains, and six species from the peri-urban areas. The forest region had a richer collection of species com- position compared to other regions due to its moderate and humid climate, which created a proper environment for sand fly development. In current study, twelve species were captured. Species diversity and richness in this study was high compared to some other species com- position studies of sand flies in Iran. In the present study, species diversity, richness and evenness in the forest (H'= 1.53, S= 8, J'= 0.59) was higher than the mountainous area. Less diversity in mountainous area was because of the presence of the dominant species Ph. kan- delakii, and the greater value of Simpson dom- inance index (D= 0.81). Contrary to our study, Shannon and evenness indices in the study of Qom (50) Province (H'= 1.36, J= 0.62) was higher in mountaineous area, and in North Khorasan (51) Province, Shannon index (H'= 1.033) was higher and evenness was similar (J'= 0.357) in mountainous area. In accordance with this study, in Khouzesten Province, south of Iran, species diversity was higher in plain areas (48). It should be mentined that the values of Shannon biodiversity index, affected by richness, eveness and Simpson, were high in the studied areas, jointly in the months of May and June. The high diversity indices could be because of humid climate due to low distance to the Caspian Sea and suitable con- dition for sand fly breeding. Conclusion This study determined some bioecological aspects of sand flies in Sari County, Mazanda- ran Province, northern Iran. The diversity and richness of the species was high, which could be because of the moderate climate due to its geographical proximity to the Caspian Sea. According to the results of this study, leish- maniasis vectors Ph. kandelakii, Ph. papatasi and Ph. major were present in the study area. Considering Mazandaran Province as one of the sporadic foci of leishmaniasis and geograph- ical proximity of Mazandaran Province with ZCL and ZVL foci of Golestan Province in the northeast and Azerbaijan Province in the north- west, respectively, and the lack of study of dis- ease vectors in this region, health authorities are recommended to highten the surveillance system and conduct more studies on the ecol- ogy of sand flies and leishmanial infections, to prevent the emergence of a new focus of leish- maniasis. Acknowledgements This work was supported by Research Dep- uty of Mazandaran University of Medical Sci- ences, Project No. 1958. We thank the staff of health centers for their assistance in the pro- ject. Ethical considerations This project has been approved by Mazanda- ran University of Medical Sciences (MUMS) ethics committee and has been registered with the code IR.MAZUMS..REC.140011513. Conflicts of Interest Authors declare that there is no conflict of interest. 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