Diversity of Sand Flies (Diptera: Psychodidae) in Endemic Focus of Visceral Leishmaniasis in Azar Shahr District, East Azarbaijan Province, North West of Iran.

BACKGROUND: There are nearly 1000 species of Phlebotomine sand flies in 6 genera, of which only two, Phlebotomus in the old world and Lutzomyia in the new world are medically important. Globally, leishmaniasis prevalent in 98 countries and affects estimated 12 million people with almost two million new cases per year. Some rural areas of Azarshahr District in East Azarbaijan Province have been reported to be endemic for visceral leishmaniasis. This study is the first attempt to determine the species diversity and density in a new focus of visceral leishmaniasis in Azarshahr District, East Azarbaijan Province, Iran.
METHODS: Sand flies were collected form indoor and outdoor biweekly using sticky traps. Diversity index of the collected sand flies within different villages were estimated by the Shannon-Weaver.
RESULTS: The activity of the sand flies extended from April to October with one peak in August. Diversity of sand flies within study area were estimated as 0.917, 1.867, 1.339, 1.673, and 1.562 in Almalodash, Jaragil, Segaiesh, Amirdizaj, and Germezgol Vvillages, respectively.
CONCLUSION: Identifying the diversity and seasonal abundance of the collected species is of importance for prediction of the period of maximum risk for leishmaniasis transmission and for the successful implementation of a control program. Species diversity is one of the most important factors in ecological studies.

Introduction

Phlebotomine sand flies belong to the family Psychodidae (Young and Duncan 1994). Psychodidae family is characterized by numerous parallel veins running to wing margin and the presence of dense hairs on the wings and thorax. From 5 Psychodidae subfamilies, Phlebotominae is known to Phlebotomine sand flies by biting mouth parts that are longer than the head, five-segmented palps, nearly cylindrical antennae, a five-brached radial vein on the wing, and the absence of an eye-bridge (Lane and Crosskey 1993). Generally, sand flies are distinguished from other small flies by their size (2.5–3.5mm in length), characteristic hopping flight, and the “V” position in which they hold their wings while resting (Triplehom and Jhonson 2005).

In the old world, about 40 species of Phlebotomus have been proven or suspected vectors of Leishmania (Rassi 2004). In Iran, 44 species of the sand flies are found and the presence of other 10 species is doubtful. In the endemic center of northwest of Iran, two species of Ph. kandelakii and Ph. Perfiliewi and in the south of Iran, three species of, Ph. keshishiani, Ph. alexandri and Ph. major are the vectors of the disease (Rassi 2007, Azizi 2009).

Leishmaniasis is prevalent in 98 countries. This parasitic disease is known one of ten major diseases by WHO (Maia et al. 2011). It has widely spread not only in tropical countries but also in some regions with very different topography (Faiman 2000, WHO 2010). Clinically, leishmaniasis is observed in four types: Visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), mucocutaneus leishmaniasis (ML) and difuse cutaneous leishmaniasis (DCL) (WHO 1994). From 4 mentioned types, only two types of visceral and cutaneous leishmaniasis are common in Iran. Visceral leishmaniasis is considered as the main problems in Iran (Rassi 2011). This disease is observed in all areas of Iran sporadically but the endemic foci of the disease have been reported from Ardabil, Fars, Boshehr and East Azerbaijan Provinces (Rassi et al. 2005, Oshaghi et al. 2009, Rassi et al. 2010).

Some rural areas of Azarshar District in East Azarbaijan Province have been reported to be endemic for visceral leishmaniasis with more than 34 new cases. Performing the control plans without sufficient information about the fauna and ecological parameters is not possible. Therefore, this study was carried out during late April to late October 2010 in rural areas of Azarshahr District, East Azarbaijan Province, Northwestern Iran, to identify diversity, species composition and other ecological parameters.

Material and Methods

Study area

Azar Shahr is located 45 km far from Tabriz. The geographical coordinates is 45 30′E and 37°30′N. The height of this county from the sea level is 1368 meters. It is limited to Lake of Urmia from the west, to Ajabshir City from the south, to Osko from the north and to Sahand Mountains from the east. In this county, the average rainfall and the relative humidity were 303 and 49 millimeters, respectively. The mean of the annual temperature is 15.25 and the mean of monthly maxi-mum and minimum temperatures were 27.7 and 3.7 °C respectively. The population of this county has been 103952 people in 2006.

Selection of Study villages

According to reported cases of the disease, a number of villages including: Segaiesh, Amirdizaj, Almalodash, Jaragil and Germezgol were selected from the county of Azarshahr in east Azarbaijan Province.

Segaiesh village: It is located 14 km from Azar Shahr with 183 families and 633 people in 2010.

Amirdizaj Village: it is located 16 km from Azar Shahr with 250 families and 1046 people.

Almalodash Village: It is located 20 km from Azar Shahr with 182 family and 860 people.

Jaragil Village: It is located 15 km from Azar Shahr with 110 families and 422 people.

Germezgol Village: It is 12 km from Azar Shahr with 70 families and 300 people in 2010.

Sand fly sampling

Sand flies were collected using sticky traps (castor oil coated white papers 20 cm× 30 cm) biweekly from selected villages indoors (bedrooms, guest room, toilet and stable) and outdoors (rodent burrow). Traps (60 papers per village) were installed in sunset and sand flies were collected during sunrise. Sample collection began in late April and continued until late October 2010, when sand fly activities were reduced sharply. Collected sand flies were stored in 96% ethanol. In the laboratory for species identification, head and three last abdomen segments of females, head with whole body of males specimens were mounted in Puri’s medium. Species identification were carried out after one week using keys of sand flies (Rassi and Hanafi bojd 2006).

Determination of Density

The density of sand flies collected by sticky traps was calculated using the formula number of specimens / M2 of sticky traps and numbers of specimens/number of traps. Logistic regression used to investigate the existence of a correlation between the abundance of the sand flies, and average monthly temperature in the study area using SAS software (Kasap et al. 2009, Tarallo 2009).

The density of species was corrected according to the formula (Guernaoui 2009, Rajider 2010)

Determination of the Species Diversity

To calculate the species diversity in a population, there are different methods but in most resources, Shannon-Weaver index has been used. Shannon-Weaver index is shown as . In this formula, S is the Richness of the species and pi is the proportion of species Ith in the population and log e is common logarithm. To convert it to common logarithm, it should be multiply in 2.303. In the present study, it is used Shannon-Weaver formula (H').

Species diversity composed of two factors, species richness and species evenness. There are many kind of species diversity indexes, a common one is the Shannon-Weaver index:

The diversity index

The proportion of the Ith species

Natural log

Number of species in the community

Results

According to formula, monthly average density, number of specimens/m2 of sticky traps were 37.60, 41.10, 40.23, 30.38 and 30.67 in Almalodash, Jaragil, Segaieesh, Amirdizaj and Germezgol, respectively. Corrected monthly average density, number of specimens/number of traps, in mentioned villages were 1.47, 1.51, 1.39 and 1.40.

Discussion

This study is the report of the results of entomology investigations on sand flies in Azarshahr District. The geographical location of this district is appropriate for the population being potentially at risk for visceral leishmaniasis.

According to Table 1, the corrected density of sand flies in Jaragil and Segaiesh villages had highest level (1.51) and we found the lowest level (1.39) in Amirdizaj village. The monthly activities of sand flies started from first half of June and have been continued to second half of October.

Table 1.

Density of sand flies in studied villages in Azarshahr District, Eastern Azarbaijan, Iran, 2010

Village	Mean density = Number of SpecimensNumber of traps	Mean density = Number of SpecimensM^2 of traps	Mean density =1+Number of SpecimensNumber of traps
Almalodash	1.18	37.60	1.47
Amirdizaj	0.95	30.38	1.39
Jaragil	1.29	41.10	1.51
Segaiesh	1.26	40.23	1.51
Germezgol	0.96	30.67	1.4

To calculate the species diversity, we used the formula Shannon-Weaver because it has been used in most resources to calculate diversity. The results are shown in tables 2, 3, 4, 5 and 6.

Table 2.

Diversity of sand flies in Almalodash Village in Azarshahr District, Eastern Azarbaijan, Iran, 2010

Community A (Almalodash)
Species	Number	Proportion (Pi)	Loge pi	Piloge pi
Ph. papatasi	554	0.54	−.666	−.333
Ph.sergenti	77	0.08	−2.526	−.202
Ph.kandelakii	58	0.06	−2.514	−.169
Ph.(Adlerius) spp	9	0.01	−4.606	−.046
Ph.C.group	203	0.19	−1.661	−.316
Ph.halepensis	78	0.07	−2.650	−.186
Ph.longiductus	19	0.02	−3.913	−.078
Ph.major	19	0.02	−3.913	−.078
Ph.mongolensis	9	0.01	−4.606	−.046
Total	1026	H'=−∑i=1s=Pi Loge pi = 1/454

Table 3.

Diversity of sand flies in Amirdizaj Village in Azarshahr District, Eastern Azarbaijan, Iran, 2010

Community B (Amirdizaj)
Species	Number	Proportion (Pi)	Loge pi	Piloge pi
Ph. papatasi	214	0.211	−1.556	−.328
Ph.sergenti	232	0.229	−1.474	−.337
Ph.mesghali	9	0.009	−1.744	−.442
Ph.c.group	175	0.173	−1.754	−.303
Ph.mongolensis	145	0.143	−1.945	−.278
Ph.tobbi	145	0.143	−1.945	−.278
Ph.kandelakii	9	0.009	−0.411	−.042
Ph.halepensis	19	0.018	−4.081	−.072
Ph.andrejevi	39	0.038	−3.271	−.124
Ph.caucasicus	19	0.018	−4.018	−.072
Ph.pawlowskyi	9	0.009	−4.711	−.042
Total	1015	H'=−∑i=1s=Pi Loge pi = 1/918

Table 4.

Diversity of sand flies in Jaragil Village in Azarshahr District, Eastern Azarbaijan, Iran, 2010

Community C (Jaragil)
Species	Number	Proportion (Pi)	Loge pi	Piloge pi
Ph. papatasi	67	0.005	−2.901	−.159
Ph.sergenti	378	0.313	−1.126	−.363
Ph.tobbi	28	0.023	−3.733	−.087
Ph.kandelakii	19	0.016	−4.136	−.006
Ph.major	9	0.007	−4.963	−.035
Ph.c.group	369	0.305	−1.187	−.362
Ph.hodsoni	39	0.032	−3.343	−.110
Ph.mongolensis	184	0.152	−1.884	−.286
Ph.halepensis	97	0.080	−2.252	−.202
Ph.longiductus	9	0.007	−4.963	−.035
Ph.andrejevi	9	0.007	−4.963	−.035
Total	1208	H'=−∑i=1s=Pi Loge pi = 1/74

Table 5.

Diversity of sand flies in Segaiesh Village in Azarshahr District, Eastern Azarbaijan, Iran, 2010

Community D (Segaiesh)
Species	Number	Proportion (Pi)	Loge pi	Piloge pi
Ph. papatasi	438	0.540	−0.616	−.333
Ph.tobbi	9	0.011	−4.514	−.049
Ph.sergenti	38	0.047	−3.058	−.144
Ph.c.group	194	0.239	−1.432	−.342
Ph.hodsoni	9	0.011	−4.511	−.049
Ph.kandelakii	38	0.047	−3.05	−.144
Ph.mongolensis	48	0.059	−2.831	−.167
Ph.halepensis	19	0.023	−3.733	−.167
Ph.perfiliewi	9	0.011	−4.511	−.049
Ph.major	9	0.011	−4.511	−.049
Total	811	H'=−∑i=1s=Pi Loge pi = 1/413

Table 6.

Diversity of sand flies in Germezgol Village in Azarshahr District, Eastern Azarbaijan, Iran, 2010

Community E (Germezigol)
Species	Number	Proportion (Pi)	Loge pi	Piloge pi
Ph. sergenti	156	0.269	−1.313	−.353
Ph.ansari	9	0.016	−4.136	−.066
Ph.kandelakii	9	0.016	−4.136	−.066
Ph.c.group	9	0.016	−4.136	−.066
Ph.halepensis	48	0.083	−2.248	−.207
Ph.mongolensis	223	0.385	−.955	−.368
Ph.tobbi	19	0.033	−3.412	−.113
Ph.andrejevi	68	0.117	−2.146	−.251
Ph.caucasicus	29	0.050	−2.996	−.149
Ph.perfiliewi	9	0.016	−4.136	−.066
Total	811	H'=−∑i=1s=Pi Loge pi = 1/413

Due to the tables of species diversity, it is obvious that the species diversity in Amirdizaj Village is in highest level than other villages and the diversity of the sand flies in this village (Population B) has more diversity and better results.

The most level of density was related to August. Whereas, there was a peak of temperature in this month. In another words, the population of the sand flies dynamically was direct correlation to the temperature of the area but to the humidity. Given the history of the emergence of the sand flies adults in the region (20 May, 2010) and the density peak of in this region, it can be determined the control programs. Especially, if there is chemical control program, we can determine exact date of residual spraying reasonably and avoid unreasonable residual spraying, further costs and extreme residual spraying.

Among 16 species collected and identified, Ph. ansarii and Ph. mesghali had lowest density (0.18%). According to the studies of Rassi et al. 2000 on the fauna of the sand flies in west north of Iran and Kaverizadeh et al. 2000 on the fauna of sand flies in Ahar, all 16 species have been reported first time in Azarshahr. Furthermore, two species of S. hodgsoni and P. mesghali have been observed in East Azerbaijan.

Two main factors of species diversity are: Richness of species and Evenness of species. Richness of species is the species diversity in the population and the Evenness of species is the species diversity of the people in the species.

The results are shown in tables 2, 3, 4, 5 and 6. In Al-Baha Province in the Saudi Arabia, a similar study has been performed (DohaandSomy 2010) with bionomics relation of Phlebotomine sand flies population with temperature and species diversity in different places that its result is compatible to our results of this study.

Conclusion

According to the results, most species diversity was observed in Amirdizaj, therefore, this village can be the best choice for determine of species composition of sand flies in the study area. It should be mentioned, identifying the diversity and seasonal abundance of the collected species is very important for prediction of the period of maximum risk for leishmaniasis transmission and for the successful implementation of a control program.