Abstract
Background:
Golestan National Park is the first, oldest and most vast national park in Iran. It was registered as a biosphere reserve by UNESCO in 1977. The park is located in Golestan, North Khorasan and Semnan Provinces. There is no information about the mosquitoes (Diptera: Culicidae) in this park.
Methods:
The larvae of mosquitoes were captured from various habitats using the standard 350 ml capacity dippers and also by pipettes, for small larval breeding sites, and buckets, for wells, during spring–autumn 2019. Larvae were preserved in lactophenol and mounted on microscope slides in Berlese medium and identified by morphological characters. The altitude and coordinates of the sampling localities, larval habitat characteristics and physicochemical features of habitat waters were recorded. Association and affinity indices were calculated.
Results:
In total, 1349 larvae including 13 species across four genera were collected: Anopheles claviger, An. maculipennis s.l., An. moghulensis, An. superpictus s.l., Culex hortensis, Cx. perexiguus, Cx. pipiens, Cx. theileri, Cx. torrentium, Cx. tritaeniorhynchus, Culiseta longiareolata, Cs. subochrea and Uranotaenia unguiculata. Anopheles moghulensis and Cx. torrentium were new to the region. Culiseta longiareolata (62.6%) displayed the most abundance. Anopheles maculipennis s.l., An. moghulensis and Cs. subochrea were the least specimens (0.1%). Larval habitat characteristics, physicochemical features of habitat waters, association occasions, and percentages were presented. The nitrate of water samples displayed a significant difference among the species (P=0.003).
Conclusion:
The study of bionomics of adult mosquitoes and detection of the vectors of different pathogens using serological or molecular-specific tests are recommended.
Keywords: Dominance, Larval habitat characteristics, Water physicochemical features, Affinity index, Association index
Introduction
Golestan National Park is the first, oldest, and most vast national park in Iran. This park has been registered as a biosphere reserve by UNESCO in 1977 (1). The plants of this park include the species from Irano-Turanian, Euro-Siberian, Mediterranean, and Saharo-Sindian floristic areas (2). This makes the park rich in flora and fauna and that is why Golestan National Park includes about 12% of the plant species, 19% of the vascular plant species, 33% of the avian species, and more than 50% of the mammal species of Iran (1, 2).
Although there are many documents about the diversity of mammals, amphibians, reptiles, birds, fishes, plants and fungi of Golestan National Park, a few data have been published about the arthropods of the park (1). As the authors know, a few published data on arthropods in this park are about Coleoptera (3), Collembola (4), Crustacea (5), Hemiptera (6, 7), Hymenoptera (8, 9), Lepidoptera (1, 10) and ticks (11), but there is no information about the mosquitoes (Diptera: Culicidae) of this biosphere reserve, despite of their medical and veterinary importance and probable influence on the wildlife.
Mosquitoes are the vectors of various protozoal, arboviral and filarial pathogens of humans and different animals. For this reason, they are the most significant family of arthropods in public health (12). The family includes two subfamilies, Anophelinae and Culicinae, 11 tribes, 41 genera, and 3726 species (13). Currently, Iranian mosquitoes include 73 species across eight genera (14–17). Seven arbo-viruses (causing agents of West Nile fever, Sindbis fever, bovine ephemeral fever, avian pox, Chikungunya fever, Rift Valley fever and dengue fever), two bacteria (causing pathogens of anthrax and tularaemia), four helminths (causal parasites of dirofilariasis, setariasis, dipetalonemiasis and lymphatic filariasis) and two protozoa (parasites of human and bird malaria) have been recorded in Iran. These pathogens are biologically or mechanically known or assumed to be vectored by mosquitoes (14, 18).
There are many published data on the larval breeding site characteristics of mosquitoes in Iran (19–35), however, the information on the physicochemical features of larval water samples and their exact correlation with the mosquito species presence and/or abundance is not very much (25, 32, 36–38). Also, there are a few articles that studied the affinity and association indices among Iranian mosquito species (24, 25, 28, 32, 39).
Basic knowledge about the fauna and ecology of the vectors is necessary to decrease the burdens of the diseases caused by agents that they transmit, applying the integrated vector management based on the One Health approach (14, 18). There is no such data about mosquitoes in Golestan National Park, despite their importance (14). To study the diversity of mosquitoes in Golestan National Park and their larval ecology, this research was carried out during 2019.
Materials and Methods
Study area
The surface area of Golestan National Park is about 91895 hectares and is located between 37°16′ 34″–37°31′00″N and 55°43′00″– 56°17′45″E. The elevation of the park ranges between 380 and 2410 meters. This park is located in three provinces of Golestan, North Khorasan and Semnan; however, the official administration and management of the park is in the Department of the Environment of Golestan Province (Fig. 1) (1).
Fig. 1.
Topographical map of Golestan National Park including collection sites, adopted from Akhani (1998)
Sampling and morphological identification
The larvae of mosquitoes were captured from various natural and artificial oviposition sites using standard 350 ml capacity dippers and also by pipettes, for small habitats, and buckets, for wells (40). The samplings were carried out through six rounds of collections during spring–autumn 2019. Only third- and fourth-instar larvae were identified. Larvae were preserved in lactophenol and mounted on microscope slides in Berlese medium and identified using morphological keys (41). Latitude, longitude and altitude of the sampling localities were obtained using the Garmin eTrex 20 GPS device and recorded in the relevant forms. The altitude and coordinates of the localities are listed in Table 1.
Table 1.
Data collection of mosquito larvae for the localities in Golestan National Park, spring–autumn 2019
| Locality/Station | Topography | Coordinates | Altitude (m) |
|---|---|---|---|
| Soulgurd | Plain | N 37° 26′ 58″, E 56° 08′ 22″ | 1186 |
| Degirmanli | Plain | N 37° 25′ 29″, E 56° 08′ 48″ | 1289 |
| Dareh-Zoghali | Plain | N 37° 24′ 53″, E 56° 09′ 40″ | 1423 |
| Bidak | Foothill forest | N 37° 16′ 99″, E 55° 53′ 76″ | 1295 |
| Alongside Dasht-e-Shad | Foothill forest | N 37° 16′ 76″, E 55° 50′ 30″ | 1374 |
| Dasht-e-Shad | Foothill forest | N 37° 18′ 30″, E 55° 49′ 84″ | 1470 |
| After tunnel | Foothill forest | N 37° 21′ 01″, E 56° 5′ 22″ | 872 |
| After Tangrah | Foothill forest | N 37° 23′ 66″, E 55° 44′ 92″ | 412 |
| Zav | Forest | N 37° 31′ 33″, E 55° 47′ 07″ | 601 |
| Beilidagh1 | Forest | N 37° 30′ 34″, E 55° 50′ 09″ | 852 |
| Beilidagh2 | Forest | N 37° 30′ 02″, E 55° 46′ 21″ | 1339 |
| Koeilar | Forest | N 37° 30′ 43″, E 55° 50′ 45″ | 1329 |
| Shor Cheshmeh | Plain | N 37° 30′ 37″, E 55° 54′ 38″ | 1249 |
| Ghare Ghovagh | Plain | N 37° 31′ 11″, E 55° 55′ 23″ | 1147 |
| Olang Cheshmeh | Forest | N 37° 24′ 09″, E 56° 00′ 01″ | 2068 |
| Ghajar Cheshmeh | Forest | N 37° 24′ 08″, E 56° 00′ 00″ | 2072 |
| Olang Cheshmeh alongside | Foothill | N 37° 31′ 13″, E 55° 58′ 13″ | 1136 |
| Ghoshe Cheshmeh | Forest | N 37° 26′ 59″, E 55° 45′ 09″ | 1033 |
| Mirza Baylu | Plain | N 37° 21′ 36″, E 56° 15′ 20″ | 1299 |
Measure of dominance
The measure of the dominant structure of a species is defined as the percentage of the species specimens in the whole sample. The following five categories are used as measures (percentage) of eudominat (ED) species (>30%), dominant (D; 10–30%), subdominant (SD; 5–10%), recedent (R; 1–5%) and subrecedent (SR; <1%) (42, 43).
Indices of affinity and association between species
The indices of association between pairs of species were calculated using two formulae: Fager’s index of affinity and the index of association (44, 45). Fager’s index of affinity is defined by IAB=2J/nA+nB where J=number of joint occurrences, nA=total number of occurrences of species A and nB=total number of occurrences of species B. The significance of the index was analyzed using a t-test (at a 5% probability level). The index of association is defined by I=2[J/A+B–0.5] where J=the number of individuals of both species in samples where they occur together, A=the total number of individuals of species A in all samples and B=the total number of individuals of species B in all samples. In this formula, the numbers of species individuals are taken into account. The formula has a range of +1 to −1.
Larval habitat characteristics
Surface area (m2), habitat situation (permanent or transient, still or running), habitat kind (natural or artificial), vegetation situation (with or without vegetation), bottom type (mud, sand, gravel, cement or plastic), sunlight situation (full or partial sunlight or shaded) and water turbidity (clear or turbid) were recorded.
Physicochemical analysis of water of larval habitats
For analysis of physicochemical characteristics, water samples were collected from different habitats in 1000 ml polyethylene bottles and transferred to the laboratory with cold boxes. The samples were analyzed for turbidity (NTU), acidity (pH), water temperature (°C), total dissolved solids (TDS) (mg/l), electrical conductivity (EC) (μS/cm), total hardness (mg/l), chemical oxygen demand (COD) (mg/l) and ions such as bicarbonate (HCO3), calcium (Ca), carbonate (CO3), chloride (Cl), fluorine (F), magnesium (Mg), nitrate (NO3), nitrite (NO2) and sulphate (SO4). The analysis of physicochemical characteristics was carried out in the Vice-Chancellorship for Health, North Khorasan University of Medical Sciences. The physicochemical features of water samples were measured according to standard methods (46).
Statistical analysis
The normality assumption of physicochemical parameters of water samples was compared by the Shapiro-Wilk test of normality. The means physicochemical parameters of water samples among different species were compared by one-way analysis of variance (ANOVA) or Kruskal-Wallis test, as appropriate. Data were analyzed using SPSS software (Version 21 for Windows, SPSS Inc., Chicago, IL).
Results
In total, 1349 specimens including 13 species across four genera of mosquitoes were captured from 38 larval habitats and identified as: Anopheles claviger, An. maculipennis s.l., An. moghulensis, An. superpictus s.l., Culex hortensis, Cx. perexiguus, Cx. pipiens, Cx. theileri, Cx. torrentium, Cx. tritaeniorhynchus, Culiseta longiareolata, Cs. subochrea and Uranotaenia unguiculata. The occurrences of An. moghulensis and Cx. torrentium were new to the region. Culiseta longiareolata (62.6%) displayed the most abundance and was eudominant. Anopheles maculipennis s.l., An. moghulensis, Cs. subochrea as well as Cx. tritaeniorhynchus were captured with the least abundance (Subrecedent) (Table 2). Culiseta longiareolata, An. claviger and Cx. hortensis were collected more than other species from different larval habitats including 25, 16 and 13 occasions, while An. moghulensis and Cs. subochrea (each with one occasion) and An. maculipennis s.l., Cx. perexiguus and Cx. tritaeniorhynchus (each with two occasions) displayed the least collection occasions (larval breeding sites) (Table 3). The association percentages of larvae are shown in Table 4. Culiseta longiareolata larvae were captured alone (22.8%) more than any other species (Table 4). The indices of affinity and association of larvae are displayed in Table 5.
Table 2.
Composition, abundance and dominance structure of mosquito larvae in Golestan National Park, spring–autumn 2019
| Species | n | % | Dominance |
|---|---|---|---|
| Anopheles claviger | 136 | 10.1 | Dominant |
| An. maculipennis s.l. | 2 | 0.1 | Subrecedent |
| An. moghulensis | 1 | 0.1 | Subrecedent |
| An. superpictus s.l. | 40 | 2.9 | Recedent |
| Culex hortensis | 183 | 13.6 | Dominant |
| Cx. perexiguus | 36 | 2.7 | Recedent |
| Cx. pipiens | 28 | 2.1 | Recedent |
| Cx. theileri | 21 | 1.5 | Recedent |
| Cx. torrentium | 24 | 1.8 | Recedent |
| Cx. tritaeniorhynchus | 8 | 0.6 | Subrecedent |
| Culiseta longiareolata | 845 | 62.6 | Eudominant |
| Cs. subochrea | 1 | 0.1 | Subrecedent |
| Uranotaenia unguiculata | 24 | 1.8 | Recedent |
| Total | 1349 | 100 |
Table 3.
Association occasions of mosquito larvae in Golestan National Park, spring–autumn 2019
| Species | Total occasions (out of 38) | Anopheles claviger | An. maculipennis s.l. | An. moghulensis | An. superpictus s.l. | Culex hortensis | Cx. perexiguus | Cx. pipiens | Cx. theileri | Cx. torrentium | Cx. tritaeniorhynchus | Culiseta longiareolata | Cs. subochrea | Uranotaenia unguiculata |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Anopheles claviger | 16 | 2 | 1 | 6 | 6 | 2 | 3 | 3 | - | 1 | 6 | 1 | 3 | |
| An. maculipennis s.l. | 2 | 2 | - | 2 | 1 | 1 | 1 | 1 | - | - | - | - | 1 | |
| An. moghulensis | 1 | 1 | - | 1 | - | 1 | - | 1 | - | 1 | - | - | - | |
| An. superpictus s.l. | 8 | 6 | 2 | 1 | 5 | 2 | 2 | 2 | 1 | 2 | 2 | - | 2 | |
| Culex hortensis | 13 | 6 | 1 | - | 5 | - | 5 | - | 1 | - | 5 | - | 1 | |
| Cx. perexiguus | 2 | 2 | 1 | 1 | 2 | - | 1 | 2 | - | 1 | - | - | 1 | |
| Cx. pipiens | 6 | 3 | 1 | - | 2 | 5 | 1 | 1 | - | - | 4 | - | 2 | |
| Cx. theileri | 4 | 3 | 1 | 1 | 2 | - | 2 | 1 | 1 | 1 | 1 | - | 2 | |
| Cx. torrentium | 4 | - | - | - | 1 | 1 | - | - | 1 | - | 3 | - | 1 | |
| Cx. tritaeniorhynchus | 2 | 1 | - | 1 | 2 | - | 1 | 1 | - | - | - | - | ||
| Culiseta longiareolata | 25 | 6 | - | - | 2 | 5 | - | 4 | 1 | 3 | - | 1 | - | |
| Cs. subochrea | 1 | 1 | - | - | - | - | - | - | - | - | - | 1 | - | |
| Uranotaenia unguiculata | 3 | 3 | 1 | - | 2 | 1 | 1 | 2 | 2 | 1 | - | - | - |
Table 4.
Association percentages of mosquito larvae in Golestan National Park, spring–autumn 2019
| Species association | Abundance (%) |
|---|---|
| Anopheles claviger | 136 (100) |
| Cx. hortensis, Cx. pipiens, Ur. unguiculata | 42 (31.0) |
| Cs. longiareolata, Cs. subochrea | 25 (18.4) |
| Cs. longiareolata | 19 (14.0) |
| Alone | 15 (11.0) |
| An. maculipennis s.l., An. superpictus, Cx. hortensis | 12 (8.9) |
| An. superpictus, Cx. hortensis | 7 (5.1) |
| Cx. hortensis | 7 (5.1) |
| Cx. theileri | 4 (3.0) |
| An. maculipennis s.l., An. superpictus, Cx. perexiguus, Cx. pipiens, Cx. theileri, Ur. unguiculata | 2 (1.4) |
| An. superpictus, An. moghulensis, Cx. perexiguus, Cx. theileri, Cx. tritaeniorhynchus | 2 (1.4) |
| Cx. pipiens, Cs. longiareolata | 1 (0.7) |
| Anopheles maculipennis s.l. | 2 (100) |
| An. claviger, An. superpictus, Cx. perexiguus, Cx. pipiens, Cx. theileri, Ur. unguiculata | 1 (50) |
| An. claviger, An. superpictus, Cx. hortensis | 1 (50) |
| Anopheles moghulensis | 1 (100) |
| An. claviger, An. superpictus, Cx. perexiguus, Cx. theileri, Cx. tritaeniorhynchus | |
| Anopheles superpictus s.l. | 40 (100) |
| An. claviger, An. maculipennis s.l., Cx. hortensis | 14 (35.0) |
| An. claviger, An. moghulensis, Cx. perexiguus, Cx. theileri, Cx. tritaeniorhynchus | 7 (17.5) |
| An. claviger, Cx. hortensis | 7 (17.5) |
| Cx. tritaeniorhynchus | 7 (17.5) |
| An. claviger, An. maculipennis s.l., Cx. perexiguus, Cx. pipiens, Cx. theileri, Ur. unguiculata | 3 (7.5) |
| Cx. hortensis, Cx. pipiens, Cs. longiareolata | 1 (2.5) |
| Cx. hortensis, Cx. torrentium, Cs. longiareolata | 1 (2.5) |
| Culex hortensis | 183 (100) |
| An. superpictus, Cx. pipiens, Cs. longiareolata | 82 (44.8) |
| An. claviger | 41 (22.4) |
| An. claviger, An. maculipennis s.l., An. superpictus | 21 (11.5) |
| Cx. pipiens, Cs. longiareolata | 10 (5.5) |
| Cx. pipiens | 10 (5.5) |
| An. claviger, An. superpictus | 7 (3.8) |
| An. claviger, Cx. pipiens, Ur. unguiculata | 4 (2.2) |
| Alone | 4 (2.2) |
| Cs. longiareolata | 3 (1.6) |
| An. superpictus, Cx. torrentium, Cs. longiareolata | 1 (0.5) |
|
| |
| Culex perexiguus | 36 (100) |
| An. claviger, An. maculipennis s.l., An. superpictus, Cx. pipiens, Cx. theileri, Ur. unguiculata | 34 (94.4) |
| An. claviger, An. moghulensis, An. superpictus, Cx. theileri, Cx. tritaeniorhynchus | 2 (5.6) |
| Culex pipiens | 28 (100) |
| Cx. hortensis | 8 (28.6) |
| Cx. hortensis, Cs. longiareolata | 7 (25.0) |
| An. claviger, An. maculipennis s.l., An. superpictus, Cx. perexiguus, Cx. theileri, Ur. unguiculata | 4 (14.3) |
| An. claviger, Cx. hortensis, Ur. unguiculata | 3 (10.7) |
| An. superpictus, Cx. hortensis, Cs. longiareolata | 3 (10.7) |
| An. claviger, Cs. longiareolata | 3 (10.7) |
| Culex theileri | 21 (100) |
| An. claviger, An. maculipennis s.l., An. superpictus, Cx. perexiguus, Cx. pipiens, Ur. unguiculata | 14 (66.6) |
| An. claviger, An. moghulensis, An. superpictus, Cx. perexiguus, Cx. tritaeniorhynchus | 4 (19.0) |
| Cx. torrentium, Ur. unguiculata | 1 (4.8) |
| An. claviger | 1 (4.8) |
| Cs. longiareolata | 1 (4.8) |
| Culex torrentium | 24 (100) |
| Cx. theileri, Ur. unguiculata | 13 (54.2) |
| Cs. longiareolata | 9 (37.5) |
| An. superpictus, Cx. hortensis, Cs. longiareolata | 2 (8.3) |
| Culex tritaeniorhynchus | 8 (100) |
| An. superpictus | 6 (75) |
| An. claviger, An. moghulensis, An. superpictus, Cx. perexiguus, Cx. theileri | 2 (25) |
| Culiseta longiareolata | 845 (100) |
| Alone | 193 (22.8) |
| An. superpictus, Cx. hortensis, Cx. pipiens | 177 (20.9) |
| Cx. theileri | 165 (19.5) |
| Cx. hortensis, Cx. pipiens | 143 (16.9) |
| An. superpictus, Cx. hortensis, Cx. torrentium | 58 (6.9) |
| An. claviger, Cs. subochrea | 52 (6.2) |
| An. claviger | 33 (3.9) |
| Cx. torrentium | 11 (1.3) |
| Cx. hortensis | 9 (1.1) |
| An. claviger, Cx. pipiens | 4 (0.5) |
| Culiseta subochrea | 1 (100) |
| An. claviger, Cs. longiareolata | |
| Uranotaenia unguiculata | 24 (100) |
| An. claviger, An. maculipennis s.l., An. superpictus, Cx. perexiguus, Cx. pipiens, Cx. theileri | 12 (50) |
| An. claviger, Cx. hortensis, Cx. pipiens | 11 (45.8) |
| Cx. theileri, Cx. torrentium | 1 (4.2) |
Table 5.
Indices of affinity and association of mosquito larvae in Golestan National Park, spring–autumn 2019. The lower part shows the index of association. The upper part shows Fager’s index of affinity. For bold numbers (significant affinities and associations), see the discussion.
|
Fager’s Index of Affinity
Index of Association |
An. claviger | An. maculipennis s.l. | An. moghulensis | An. superpictus s.l. | Culex hortensis | Cx. perexiguus | Cx. pipiens | Cx. theileri | Cx. torrentium | Cx. tritaenio rhynchus | Cs. longiareolata | Cs. subochrea | Ur. unguiculata |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Anopheles claviger | 0.222 | 0.117 | 0.500 | 0.413 | 0.222 | 0.272 | 0.300 | - | 0.111 | 0.292 | 0.117 | 0.315 | |
| An. maculipennis s.l. | −0.78 | - | 0.400 | 0.133 | 0.500 | 0.250 | 0.333 | - | - | - | - | 0.400 | |
| An. moghulensis | −0.96 | - | 0.222 | - | 0.666 | - | 0.400 | - | 0.500 | - | - | - | |
| An. superpictus s.l. | −0.40 | 0.22 | −0.62 | 0.476 | 0.400 | 0.285 | 0.333 | 0.333 | 0.400 | 0.121 | - | 0.363 | |
| Culex hortensis | −0.12 | −0.78 | - | 0.20 | - | 0.526 | - | 0.117 | - | 0.263 | - | 0.125 | |
| Cx. perexiguus | −0.54 | 0.84 | −0.84 | 0.20 | - | 0.250 | 0.666 | - | 0.500 | - | - | 0.400 | |
| Cx. pipiens | −0.34 | −0.68 | - | −0.68 | 0.20 | 0.18 | 0.200 | - | - | 0.258 | - | 0.444 | |
| Cx. theileri | −0.66 | 0.30 | −0.56 | −0.10 | - | 0.88 | −0.28 | 0.250 | 0.333 | 0.068 | - | 0.571 | |
| Cx. torrentium | - | - | - | −0.92 | −0.98 | - | - | −0.38 | - | 0.206 | - | 0.285 | |
| Cx. tritaeniorhynchus | −0.96 | - | −0.34 | −0.10 | - | −0.82 | - | −0.60 | - | - | - | - | |
| Culiseta longiareolata | −0.74 | - | - | −0.48 | −0.08 | - | −0.24 | −0.62 | −0.82 | - | 0.076 | - | |
| Cs. subochrea | −0.64 | - | - | - | - | - | - | - | - | - | −0.88 | - | |
| Uranotaenia unguiculata | −0.18 | 0.00 | - | −0.54 | −0.86 | 0.52 | 0.14 | 0.24 | −0.42 | - | - | - |
The significant affinities and associations of species pairs were displayed with bold numbers (also see discussion). The larval habitat characteristics and occurrence percentages of larvae are shown in Table 6. The surface area of most larval habitats was small (about 1–10 m2, in one case about 25 m2). All larvae were collected from clear (versus turbid) habitats. Ten species (out of 13) were mostly collected from natural habitats (75.8–100%). Anopheles moghulensis and Cx. tritaeniorhynchus were collected only from wells (Artificial habitats). Also, about 44% of Cs. longiareolata larvae were collected from artificial oviposition sites (Table 6). The physicochemical parameters of the water samples of larval habitats are shown in Table 7. The normality assumption was not met for any of the parameters (P<0.05), hence nonparametric analysis was used. There was no significant difference in physicochemical parameters (P>0.05) (Table 7). The physicochemical parameters of the habitat water samples of larvae for each species were displayed in Tables 8 and 9. Only nitrate displayed a significant difference among the species (P=0.003).
Table 6.
Larval habitat characteristics and occurrence percentages of mosquito larvae in Golestan National Park, spring–autumn 2019
| Larval habitat characteristics | Anopheles claviger | An. maculipennis s.l. | An. moghulensis | An. superpictus s.l. | Culex hortensis | Cx. perexiguus | Cx. pipiens | Cx. theileri | Cx. torrentium | Cx. tritaeniorhynchus | Culiseta longiareolata | Cs. subochrea | Uranotaenia unguiculata |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A) Habitat situation | |||||||||||||
| 1. Permanent | 67.5 | 50 | - | 63.6 | 31.3 | - | 10.7 | 5.0 | 91.7 | - | 2.9 | - | 50 |
| 2. Transient | 32.5 | 50 | 100 | 36.4 | 68.7 | 100 | 89.3 | 95.0 | 8.3 | 100 | 97.1 | 100 | 50 |
| 3. Slow-running water | 69.8 | 50 | 63.6 | 42.0 | 71.4 | - | - | 2.0 | 45.8 | ||||
| 4. Stagnant water | 30.2 | 50 | 100 | 36.4 | 58.0 | 100 | 28.6 | 100 | 100 | 100 | 98.0 | 100 | 54.2 |
| B) Vegetation situation | |||||||||||||
| 1. Without vegetation | 31.0 | 50 | 100 | 87.9 | 72.7 | 100 | 89.3 | 100 | 100 | 100 | 99.8 | 100 | 54.2 |
| 2. With vegetation | 69.0 | 50 | - | 12.1 | 27.3 | - | 10.7 | - | - | - | 0.2 | - | 45.8 |
| C) Substrate type | |||||||||||||
| 1. Mud | 95.2 | 100 | 100 | 84.9 | 32.3 | 100 | 57.1 | 100 | 91.7 | 100 | 16.1 | 100 | 100 |
| 2. Sand | 4.8 | - | - | 12.1 | 60.8 | - | 10.7 | - | - | - | 39.6 | - | - |
| 3. Gravel | - | - | - | - | 5.7 | - | 28.6 | - | - | - | - | - | - |
| 4. Cement | - | - | - | 3.0 | 0.6 | - | - | - | 8.3 | - | 13.0 | - | - |
| 5. Plastic | - | - | - | - | 0.6 | - | 3.6 | - | - | - | 31.3 | - | - |
| E) Sunlight situation | |||||||||||||
| 1. Full sunlight | 13.5 | 50 | 100 | 78.8 | 15.9 | 5.6 | 3.6 | 20.0 | 8.3 | 100 | 44.3 | - | - |
| 2. Partial sunlight | 25.4 | - | - | 3.0 | 80.7 | - | 71.4 | - | - | - | 41.4 | - | - |
| 3. Shaded | 61.1 | 50 | - | 18.2 | 3.4 | 94.4 | 25.0 | 80.0 | 91.7 | - | 14.3 | 100 | 100 |
| F) Habitat type | |||||||||||||
| 1. Natural habitat | |||||||||||||
| 1a. Ground pool | 20.6 | - | - | 3.0 | 46.6 | - | 21.4 | - | - | 52.1 | 100 | - | |
| 1b. River bed pool | 4.0 | - | - | 9.1 | 1.1 | - | - | - | - | - | - | - | - |
| 1c. River edge | - | - | - | - | - | - | - | - | - | - | - | - | - |
| 1d. Spring | 70.6 | 50 | - | 42.5 | 35.2 | - | 60.7 | 10.0 | 91.7 | - | 3.6 | - | 50 |
| 1e. Stream edge | 3.2 | 50 | - | 21.2 | 15.9 | 94.4 | 14.3 | 70.0 | - | - | - | - | 50 |
| 2. Artificial habitat | |||||||||||||
| 2a. Animal cement trough | - | - | - | 3.0 | 0.6 | - | - | - | 8.3 | - | 13.0 | - | - |
| 2b. Water storage tank | - | - | - | - | 0.6 | - | 3.6 | - | - | - | 31.3 | - | - |
| 2c. Well | 1.6 | - | 100 | 21.2 | - | 5.6 | - | 20.0 | - | 100 | - | - | - |
Table 7.
Physicochemical parameters of the water samples of larval habitats in Golestan National Park, spring–autumn 2019
| Parameter | Mean | Standard deviation | Minimum | Maximum | Sample Number |
|---|---|---|---|---|---|
| Turbidity (NTU) | 3.83 | 3.87 | 0.60 | 17.50 | 12 |
| Acidity (pH) | 7.42 | 0.23 | 7.03 | 8.00 | 14 |
| Temperature (°C) | 19.7 | 4.9 | 9.0 | 24.9 | 14 |
| Total dissolved solids (TDS) (mg/l) | 672.1 | 685.7 | 180.3 | 4230.0 | 14 |
| Electrical conductivity (EC) (μS/cm) | 1213.5 | 1224.1 | 304.0 | 7620.0 | 14 |
| Total hardness (CaCO3) (mg/l) | 514.0 | 464.3 | 200.0 | 3060.0 | 14 |
| Chemical oxygen demand (COD) (mg/l) | 19.6 | 19.2 | 0.0 | 70.0 | 12 |
| Fluoride (F) (mg/l) | 0.64 | 0.36 | 0.02 | 1.72 | 14 |
| Chloride (Cl) (mg/l) | 145.91 | 236.16 | 9.99 | 1399.56 | 14 |
| Sulphate (SO4) (mg/l) | 313.8 | 804.6 | 1.0 | 5382.0 | 14 |
| Carbonate (CO3) (mg/l) | 1.0 | 4.5 | 0.0 | 20.0 | 14 |
| Bicarbonate (HCO3) (mg/l) | 302.0 | 112.0 | 97.6 | 658.8 | 14 |
| Nitrite (NO2) (mg/l) | 0.007 | 0.017 | 0.000 | 0.083 | 14 |
| Nitrate (NO3) (mg/l) | 42.3 | 104.8 | 0.0 | 341.0 | 14 |
| Calcium (Ca) (mg/l) | 129.9 | 123.1 | 9.6 | 704.0 | 14 |
| Magnesium (Mg) (mg/l) | 44.1 | 44.1 | 1.6 | 312.0 | 14 |
Table 8.
Physicochemical parameters of the habitat water samples of mosquito larvae in Golestan National Park, spring–autumn 2019
| Species (Total number of occurrences) | Physicochemical parameters (Mean±SD, Minimum–Maximum, Number) | ||||||
|---|---|---|---|---|---|---|---|
|
| |||||||
| Turbidity (NTU) | Acidity (pH) | Temperature (°C) | Total dissolved solids (TDS) (mg/l) | Electrical conductivity (EC) (μS/cm) | Total hardness (CaCO3) (mg/l) | Chemical oxygen demand (COD) (mg/l) | |
| Anopheles claviger (16) | 1.93±0.97 (1.35–3.90, 6) | 7.30±0.22 (7.03–7.70, 10) | 19.2±5.2 (9.0–24.8, 10) | 515.0±324.7 (180.3–1227.0, 10) | 915.7±524.7 (304.0–2120.0, 10) | 414.0±188.2 (200.0–750.0, 10) | 11.1±8.7 (3.0–26.0, 6) |
| An. maculipennis s.l. (2) | - | 7.25 (1) | 21.2 (1) | 300.0 (1) | 570.0 (1) | 270.0 (1) | - |
| An. moghulensis (1) | 3.90 (1) | 7.7 (1) | 9.0 (1) | 1227.0 (1) | 2120.0 (1) | 750.0 (1) | 26.0 (1) |
| An. superpictus s.l. (8) | 4.00±0.14 (3.90–4.11, 2) | 7.50±0.18 (7.25–7.70, 6) | 19.7±5.4 (9.0–24.9, 6) | 665.8±576.0 (230.0–1556.0, 6) | 1208.6±1000.8 (472.0–2800.0, 6) | 476.6±349.4 (210.0–1060.0, 6) | 35.0±12.7 (26.0–44.0, 2) |
| Culex hortensis (13) | 3.09±2.35 (1.35–7.98, 7) | 7.36±0.17 (7.08–7.69, 11) | 19.6±3.8 (14.6–24.9, 11) | 494.1±398.6 (180.3–1556.0, 11) | 885.9±697.6 (304.0–2800.0, 11) | 391.8±236.9 (200.0–1060.0, 11) | 21.0±20.8 (0.0–49.0, 7) |
| Cx. perexiguus (2) | 3.90 (1) | 7.70 (1) | 9.0 (1) | 1227.0 (1) | 2120.0 (1) | 750.0 (1) | 26.0 (1) |
| Cx. pipiens (6) | 3.55±2.68 (1.55–7.98, 5) | 7.33±0.20 (7.03–7.58, 6) | 19.4±4.5 (15.0–24.9, 6) | 386.6±151.3 (230.0–661.0, 6) | 690.5±239.9 (465.0–1102.0, 6) | 390.0±173.7 (220.0–720.0, 6) | 29.6±19.2 (3.0–49.0, 5) |
| Cx. theileri (4) | 3.72±0.24 (3.55–3.90, 2) | 7.85±0.21 (7.70–8.00, 2) | 15.9±9.7 (9.0–22.8, 2) | 792.0±615.1 (357.0–1227.0, 2) | 1409.0±1005.5 (698.0–2120.0, 2) | 500.0±353.5 (250.0–750.0, 2) | 13.0±18.3 (0.0–26.0, 2) |
| Cx. torrentium (4) | - | 7.32 (1) | 21.2 (1) | 1556.0 (1) | 2800 (1) | 1060 (1) | - |
| Cx. tritaeniorhynchus (2) | 3.90 (1) | 7.62±0.10 (7.55–7.70, 2) | 15.1±8.6 (9.0–21.2, 2) | 737.5±692.2 (248.0–1227.0, 2) | 1296.5±1164.6 (473.0–2120.0, 2) | 480.0±381.8 (210.0–750.0, 2) | 26.0 (1) |
| Culiseta longiareolata (25) | 5.32±6.18 (0.60–17.50, 12) | 7.46±0.26 (7.03–8.00, 14) | 22.2±3.0 (15.0–24.9, 14) | 940.4±1156.5 (205.7–4230.0, 14) | 1728.7±2085.2 (423.0–7620.0, 14) | 720.7±817.7 (220.0–3060.0, 14) | 15.9±24.3 (0.0–70.0, 12) |
| Cs. subochrea (1) | - | 7.20 (1) | 22.4 (1) | 444 (1) | 841 (1) | 390.0 (1) | - |
| Uranotaenia unguiculata (3) | - | 7.32 (1) | 21.3 (1) | 431 (1) | 813 (1) | 380.0 (1) | - |
Table 9.
Chemical parameters (ions) of the habitat water samples of mosquito larvae in Golestan National Park, spring–autumn 2019
| Species (Total number of occurrences) | Chemical parameters (Mean±SD, Minimum–Maximum, Number) (mg/l) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
|
| |||||||||
| Fluoride (F) | Chloride (Cl) | Sulphate (SO4) | Carbonate (CO3) | Bicarbonate (HCO3) | Nitrite (NO2) | Nitrate (NO3) | Calcium (Ca) | Magnesium (Mg) | |
| Anopheles claviger (16) | 0.70±0.42 (0.28–1.72, 10) | 92.96±120.42 (19.99–399.87, 10) | 130.4±162.4 (1.0–477.0, 10) | 0 (9) | 326.9±136.0 (158.6–658.8, 10) | 0.002±0.003 (0.000–0.009, 10) | 35.8±107.2 (0.0–341.0, 10) | 100.5±59.6 (9.6–200.0, 10) | 32.0±19.6 (1.6–60.0, 10) |
| An. maculipennis s.l. (2) | 0.40 (1) | 29.99 (1) | 48.0 (1) | 0 (1) | 256.2 (1) | 0.005 (1) | 1.6 (1) | 60.0 (1) | 28.8 (1) |
| An. moghulensis (1) | 0.64 (1) | 399.87 (1) | 368.0 (1) | 0 (1) | 158.6 (1) | 0 (1) | 341 (1) | 200.0 (1) | 60.0 (1) |
| An. superpictus s.l. (8) | 0.64±0.33 (0.38–1.24, 6) | 143.28±176.88 (19.99–399.87, 6) | 271.3±427.1 (3.0–1098.0, 6) | 0 (6) | 250.1±60.6 (158.6–341.6, 6) | 0.005±0.005 (0.000–0.014, 6) | 58.1±138.5 (0.8–341.0, 6) | 114.6±83.0 (48.0–240.0, 6) | 45.6±35.4 (14.4–110.4, 6) |
| Culex hortensis (13) | 0.67±0.45 (0.28–1.72, 11) | 75.42±101.78 (19.99–339.89, 11) | 177.5±333.9 (2.0–1098.0, 11) | 0 (11) | 303.8±63.4 (195.2–390.4, 11) | 0.002±0.003 (0.000–0.008, 11) | 9.2±23.5 (0.0–80.2, 11) | 94.5±52.3 (56.0–240.0, 11) | 37.3±26.5 (14.4–110.4, 11) |
| Cx. perexiguus (2) | 0.64 (1) | 399.87 (1) | 368.0 (1) | 0 (1) | 158.6 (1) | 0 (1) | 341.0 (1) | 200.0 (1) | 60.0 (1) |
| Cx. pipiens (6) | 0.52±0.21 (.37–0.86, 6) | 38.31±24.00 (19.99–79.97, 6) | 47.3±52.8 (2.0–125.0, 6) | 0 (6) | 378.2±147.2 (231.8–658.8, 6) | 0.0006±0.001 (0.000–0.004, 6) | 15.2±31.8 (0.0–80.2, 6) | 98.6±46.5 (64.0–192.0, 6) | 34.4±16.7 (14.4–57.6, 6) |
| Cx. theileri (4) | 0.52±0.16 (0.40–0.64, 2) | 219.92±254.48 (39.98–399.87, 2) | 227.5±198.6 (87.0–368.0, 2) | 10.0±14.1 (0.0–20.0, 2) | 225.7±94.8 (158.6–292.8, 2) | 0.008±0.011 (0.000–0.016, 2) | 171.0±240.3 (1.1–341.0, 2) | 128.0±101.8 (56.0–200.0, 2) | 43.2±23.7 (26.4–60.0, 2) |
| Cx. torrentium (4) | 1.24 (1) | 339.89 (1) | 1098.0 (1) | 0 (1) | 280.6 (1) | 0.004 (1) | 1.1 (1) | 240.0 (1) | 110.4 (1) |
| Cx. tritaeniorhynchus (2) | 0.54±0.14 (0.44–0.64, 2) | 209.93±268.61 (19.99–399.87, 2) | 188.0±254.5 (8.0–368.0, 2) | 0 (2) | 195.2±51.7 (158.6–231.8, 2) | 0.007±0.009 (0.000–0.014, 2) | 171.2±240.1 (1.4–341.0, 2) | 124.0±107.4 (48.0–200.0, 2) | 40.8±27.1 (21.6–60.0, 2) |
| Culiseta longiareolata (25) | 0.61±0.42 (0.02–1.37, 14) | 237.77±402.56 (9.99–1399.56, 14) | 701.8±1517.8 (1.0–5382.0, 140 | 2.8±7.2 (0.0–20.0, 14) | 316.3±128.2 (97.6–658.8, 14) | 0.020±0.032 (0.000–0.083, 14) | 10.0±22.1 (0.0–80.2, 14) | 192.0±216.5 (52.0–704.0, 14) | 57.7±78.1 (7.2–312.0, 14) |
| Cs. subochrea (1) | 0.90 (1) | 59.98 (1) | 90.0 (1) | 0 (1) | 366.0 (1) | 0.009 (1) | 1.6 (1) | 100.0 (1) | 33.6 (1) |
| Uranotaenia unguiculata (3) | 0.86 (1) | 49.98 (1) | 89.0 (1) | 0 (1) | 341.6 (1) | 0.004 (1) | 3.0 (1) | 88.0 (1) | 38.4 (1) |
Discussion
This article presents the fauna of mosquitoes of Golestan National Park for the first time, including 13 species across four genera. Two species, An. moghulensis and Cx. torrentium, are found for the first time in the region. There are no previous records for them in Golestan, North Khorasan and Semnan Provinces where this park is located. Anopheles moghulensis is recorded only in Hormozgan Province and Sistan and Baluchistan Province in southern Iran (27, 47). Its record in northern Iran needs to be verified by collecting more specimens, especially adults. Also, it is not easy to distinguish the larvae and females of Cx. torrentium and Cx. pipiens. The most reliable characters for differentiating them are those of the male genitalia (41). Culex torrentium is recorded with certainty in Ardebil Province (48), Guilan Province (49, 50) and Mazandaran Province (30, 38, 39) in northern Iran. Other records in southern Iran, such as Bushehr (51) and Fars (52, 53) Provinces need to be verified.
Among the collected species, An. maculipennis s.l. and An. superpictus s.l. are the proven vectors of malaria parasites in Iran (14). Anopheles maculipennis and Culex theileri vector Setaria labiatopapillosa, causing agent of setariasis, and the dog heart worm Dirofilaria immitis, causing agent of dirofilariasis, respectively (48). Culex pipiens and Cx. theileri are known vectors of West Nile fever virus and probably Sindbis fever virus (54–56). Dirofilariasis and West Nile fever have been found in Golestan Province (14). Also, Bakhshi et al. (57) detected chikungunya virus in An. maculipennis s. l., Cx. tritaeniorhynchus and Cs. longiareolata in North Khorasan Province employing primers designed for the CHIKV Asian genotype, but they failed to isolate the virus and perform whole genome sequencing.
Culiseta longiareolata was the most prevalent species (Eudominant) and was collected mostly with other prevalent (Dominant) species: An. claviger and Cx. hortensis (Tables 2–4). This high association was also observed in Guilan Province of northern Iran (20). Moreover, high association of Cs. longiareolata and Cx. hortensis was observed in Chaharmahal and Bakhtiari Province (32). The high percentage of isolation of this species (22.8%) may be because of the predation behavior of Cs. longiareolata larvae against other mosquito larvae (58). This was observed in the Hormozgan Province of southern Iran (25: 36.6%), Isfahan Province of central Iran (28: 37.0%) and Kurdistan Province of western Iran (26: 96.9%), either. However, this isolation was not very high in Guilan Province (20: 10.6%). This may be because of the different climate of Guilan Province, temperate with higher precipitation, which causes more available larval habitats, or other physicochemical and biological features that influence larval population and their co-occurrences (32). It is noteworthy that the determination of the seasonal activity of the species was not among the goals of this study. However, the season of collection may influence the abundance and possibility of the collection of the species in different ways, especially via changes in temperature and precipitation (32).
Although some pairs of species displayed significant indices of affinity or association (Table 5), it is not possible to discuss them with certainty, because of the rarity of individuals of them. Only three species, An. claviger, Cx. hortensis and Cs. longiareolata, were captured in higher numbers of individuals (Eudominant and dominant). They did not show any significant affinity and association, but they had higher affinity and association indices with each other (Table 5). This is a more logical result than the significant indices of affinity and association for rare species. In a study in the Hormozgan Province of southern Iran, Hanafi-Bojd et al. (24) found that there were significant affinities between eight pairs of anopheline mosquitoes: An. culicifacies s.l./An. dthali, An. culicifacies s.l./ An. stephensi, An. culicifacies s.l./ An. turkhudi, An. culicifacies s.l./ An. moghulensis, An. dthali/An. stephensi, An. dthali/An. superpictus s.l., An. dthali/ An. moghulensis and An. superpictus s.l./ An. moghulensis. In a study in the Isfahan Province of central Iran, there was no significant affinity or association between species pairs when they occurred together except for the pair of An. dthali/ An. turkhudi that each was collected one time as one larva (28). In Mazandaran Province of northern Iran, Nikookar et al. (39) observed an association between An. hyrcanus/ An. pseudopictus, but the exact difference between these two species in the larval stage and their taxonomy needs to be investigated extensively (14, 41). In another investigation in Hormozgan Province, the pair of Cx. sinaiticus/ Cx. tritaeniorhynchus showed significant affinity using Fager and McGowan’s test and the pair of Ae. caballus/ Ae. vexans (with low abundance) displayed significant association according to the index of association (25). Omrani and Azari-Hamidian (32) found significant affinities between the following pairs of species in Chaharmahal and Bakhtiari Province: Cx. theileri/ An. maculipennis s.l., Cx. theileri/ Cx. perexiguus, An. superpictus s.l./ Cx. perexiguus, An. superpictus s.l./ Cx. theileri, Cx. perexiguus/ An. maculipennis s.l. and Cs. longiareolata/ Cx. hortensis. These affinities and associations may show similar ecological requirements of these pair species. To the best of our knowledge, there is no more data about the affinity and association indices of mosquitoes in Iran.
As expected, most of the species specimens were collected from natural habitats (75.8–100%), because of the rarity of artificial habitats, but rare larvae of An. moghulensis and Cx. tritaeniorhynchus were collected only from wells (Artificial habitat) (Table 6). The rice field mosquito Cx. tritaeniorhynchus was found mostly (80.59%) in natural larval breeding sites in the Guilan Province of northern Iran, but more than 66% of the collected specimens from artificial habitats were found in rice fields (21). Also, more than 95% of the specimens of this species were found in natural oviposition sites in the Hormozgan Province of southern Iran (25). On the other hand, Zaim (19) reported that 76 % of the specimens of the species were collected from man-made (artificial) habitats, including more than 68% from rice fields, in various areas of the country. Also, Shoraka et al. (33) found 62.7% of larvae of this species in artificial habitats, including about 10% in rice fields, in Golestan Province. Interestingly, about 44% of Cs. longiareolata larvae were captured from artificial habitats (Table 6). Also, 41.4, 50, 51.3, 52 and 88.8% of this species larvae were collected from artificial larval breeding sites in Charmahal and Bakhtiari Province (32), Kashan County of Isfahan Province (34), Mazandaran Province (30), Hamedan Province (23) and Golestan Province (31), respectively. However, the species larvae were collected from natural habitats in as much as 78% of the whole country (19), 81.5% of Hormozgan Province (35), 89 to 100% of Kurdistan Province (26, 29), and 100% of Guilan Province (20). These differences may be because of sampling and different climates which cause differences in precipitations and available habitats. In this point of view, for example, Guilan and Kurdistan Provinces with more precipitations provide much more natural ovi-position sites for mosquitoes. Also, most of the available habitats in Hormozgan Province, with an arid climate, are permanent or seasonal temporary rivers or streams (natural). Another abundant species of this investigation, Cx. hortensis, was collected 98.8% from natural habitats (Table 6). The species was also collected from natural habitats as much as 71.3–100% in Kurdistan Province (26, 29), 85% in Guilan Province (21), 93.5% in the whole country (19) and 100% in Isfahan Province (28). Another prevalent species of this study, An. claviger, was collected from natural habitats as much as 98.4% (Table 6). This species was also captured from natural habitats in Mazandaran Province as much as 77.4% (30), 97.9% in Guilan Province (22) and 100% in Kurdistan Province (26). In general, it seems, these species prefer natural habitats.
In the present investigation, among the physicochemical parameters of the habitat water samples of larvae, only nitrate displayed a significant difference among the species (Tables 8 and 9). Ghanbari et al. (36) found a significant correlation between the larvae of the following anophelines with some physicochemical features in southeastern Iran: An. culicifacies s.l. (Phosphate, EC, and calcium), An. stephensi (Nitrate), An. superpictus s.l. (Total hardness, calcium), An. turkhudi (pH, total hardness, nitrate, calcium) and An. multicolor (pH, sulphate). In two studies in Iran, there were no significant correlations between physicochemical and/or microbial features and the presence of larvae in Qom Province (37) and Hormozgan Province (25). In a study in northern Iran, Cx. pipiens larval abundance showed a significant positive correlation with EC, alkalinity, total hardness and chloride (38). In an investigation in Chaharmahal and Bakhtiari Province, pH, dissolved oxygen and temperature significantly differed between the larval breeding sites of different mosquito species (32). As far as we know, there is no more information in this regard in Iran. According to a systematic review and meta-analysis, pH, turbidity, EC, dissolved oxygen, nitrogen and phosphorus displayed a significant positive pooled correlation with mosquito presence and abundance, while alkalinity showed a nonsignificant null pooled correlation (59).
One of the limitations of the present investigation was the rarity of specimens of some species. They were collected in a few numbers; thus, it was not possible to analyze their larval habitat characteristics or physicochemical features of habitat water with certainty. The reason probably was the rarity of available larval habitats, sampling limitation or the relatively low population of some species. In this regard, several limitations of the study of mosquito larvae have been listed by Banafshi et al. (26) and Omrani and Azari-Hamidian (32). Also, the possibility of the sampling of some species larvae is low, for example, some aedine species, especially tree-hole species. Hence, adult collections using manual aspirators while landing on animal or human baits, light traps with or without CO2, BG lure traps and ovitraps may increase the possibility of finding new species in the region. Another important limitation of sampling was the inaccessibility of many localities. Most areas of the park are mountainous forests without any roads for vehicles. Thus, in this study, most sampling sites were limited to those areas which were close to the present available roads.
Conclusion
Among the identified species, there are many potential vectors of pathogens that can infect humans and animals. The investigation of the ecology of mosquitoes, especially using the collections of adults, and the detection of the exact vectors using serological or molecular-specific tests are recommended for future studies.
Acknowledgements
This article is the result of a joint project financially supported by the Guilan University of Medical Sciences, Golestan University of Medical Sciences and North Khorasan University of Medical Sciences (IR.GUMS.REC.1398.283). Dr Maryam Shakiba, Department of Epidemiology and Biostatistics, School of Health, Guilan University of Medical Sciences, Rasht, Iran, is appreciated for her comments on statistical analysis. The authors thank the management of Golestan National Park, the Department of the Environment of Golestan Province, for administrative support. Also, the authors appreciate the management and personnel of the following stations of the park: Almeh, Dasht-e-Shad, Lohandar, Mirza Baylu and Soulgurd for their cooperation in the field. The following persons who helped us while providing the research proposal are acknowledged: Dr Mohammad Reza Shirzadi, Center for Disease Control and Prevention, Ministry of Health and Medical Education, Tehran, Iran, Dr Ahmad Ali Hanafi-Bojd, Department of Vector Biology and Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, and Mr. Behzad Norouzi, Research Center of Health and Environment, School of Health, Guilan University of Medical Sciences, Rasht, Iran. The authors also would like to thank Dr Hossein Akhani, Department of Plant Sciences, School of Biology, College of Science, University of Tehran, Tehran, Iran, for permission to use his map.
Footnotes
Ethical considerations
The ethical code IR.GUMS.REC.1398.283 has been registered for this study.
Conflict of interest statement
The authors declare there is no conflict of interest.
Dedication
This article is dedicated to the late Mr Dordy-Mohammad (Freydoon) Aghatabai, one of the colleagues of the present project, who passed away during conducting this research.
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