J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 207 http://jad.tums.ac.ir Published Online: June 30, 2021 Original Article Population Fluctuations and Abundance Indices of Mosquitoes (Diptera: Culicid), as the Potential Bridge Vectors of Pathogens to Humans and Animals in Mazandaran Province, Northern Iran Seyed Hassan Nikookar1; *Mahmoud Fazeli-Dinan1; *Ahmadali Enayati2 1Department of Medical Entomology and Vector Control, Health Sciences Research Center, Addiction Institute, School of Public Health, Mazandaran University of Medical Sciences, Sari, Iran 2Department of Medical Entomology and Vector Control, School of Public Health and Health Sciences Research Center, Mazandaran University of Medical Sciences, Sari, Iran *Corresponding authors: Dr Mahmoud Fazeli-Dinan, E-mail: fazelidinan@gmail.com, Dr Ahmadali Enayati, E- mail: aenayati@mazums.ac.ir (Received 1 Dec 2020; accepted 19 May 2021) Abstract Background: Seasonal activity patterns of mosquitoes are essential as baseline knowledge to understand the transmis- sion dynamics of vector-borne diseases. This study was conducted to evaluate the monthly dynamics of the mosquito populations and their relation to meteorological factors in Mazandaran Province, north of Iran. Methods: Mosquito adults and larvae were collected from 16 counties of Mazandaran Province using different sam- pling techniques, once a month from May to December 2014. “Index of Species Abundance” (ISA) along with “Stand - ardized ISA” (SISA) was used for assessing the most abundant species of mosquitoes based on the explanations of Rob- ert and Hsi. Pearson’s correlation coefficient (R) was used to assess the relationships between the monthly population fluctuations and meteorological variables. Results: Overall, 23750 mosquitoes belonging to four genera and nineteen species were collected and identified. The highest population density of mosquitoes was in July and the lowest in May. The ISA/SISA indices for Culex pipiens were both 1 for larvae and 1.25/0.973 for adults in total catch performed in human dwellings. For Cx. tritaeniorhynchus, the ISA/SISA were 1.68/0.938 in pit shelter method. A significant positive correlation was observed between population fluctuations of Cx. tritaeniorhynchus and mean temperature (R: 0.766, P< 0.027). Conclusions: The results indicated that the mosquitoes are more active in July, and Cx. pipiens and Cx. tritaeniorhyn- chus were the most abundant species. Considering the potential of these species as vectors of numerous pathogens, con- trol programs can be planed based on their monthly activity pattern in the area. Keywords: Seasonal activity; Mosquitoes; Abundance indices; Mazandaran; Caspian Sea littoral Introduction Mosquitoes are distributed almost all over the world, except a few islands and the Ant- arctica (1). Since the earliest times, mosquito bites and habitats have been related with hu- man diseases, and mosquitoes were the first arthropods formally convicted as intermediate hosts of vertebrate parasites in 1878 (2). They are the most important arthropod taxon in med- ical entomology because of their nuisance and transmission of malaria, arboviral diseases and microfilariae (3). Mosquito-borne infectious dis- eases are known as the most commonly trans- mitted diseases by vectors in terms of mortal- ity and disability-adjusted life years (4). Ma- laria is one of the most important Anopheles- borne parasitic diseases in the world. The dis- ease has a massive burden universally, an es- timated number of cases of 229 million and 409000 deaths occurred in 2019 (5). There was also approximately 96 million cases and 1091 deaths related to dengue globally. Albeit, the global burden of Zika, chikungunya and West Copyright © 2021 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:fazelidinan@gmail.com mailto:aenayati@mazums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 208 http://jad.tums.ac.ir Published Online: June 30, 2021 Nile is not as large as malaria and dengue (4), but their impact on health system is high, es- pecially as several large outbreaks of the dis- eases occur every year and their transmission is extending to new areas (6-8). Iran is in the perspective of eliminating malaria by 2025. Although there were 1105 imported cases of malaria in 2019, no indigenous malaria cases were reported in Iran since 2018 (5). Thirty species of Anopheles have been documented from Iran (9), of which, seven species have been reported from Mazandaran Province (10). Anopheles sacharovi, An. maculipennis s.l., An. fluviatilis s.l., An. stephensi, An. su- perpictus s.l., An. dthali, and An. culicifacies s.l. are identified as the proven malaria vectors in Iran (11), while An. pulcherrimus is stated as a suspected vector (12). Recently, An. hyrca- nus and An. subpictus s.l. were revealed to be infected with Plasmodium based on molecular analysis in northern and southern Iran (13, 14). Anopheles maculipennis s.l. and An. sacharovi are known to play an important role in trans- mission of malaria in the northern parts of the country (15). Zika, dengue and chikungunya are arboviral diseases transmitted by Aedes mosquitoes, es- pecially Ae. aegypti and Ae. albopictus. There are no reports of Zika virus in Iran, while im- ported cases of dengue (16-18) and chikungu- nya (19) have lately been reported from the country. Aedes aegypti and Ae. albopictus are the main vectors of dengue, chikungunya and Zika, worldwide. Aedes aegypti had been re- ported in southern parts of Iran in 1920–1953 (20-22), however, it retreated to Arabian Pen- insula and northern Africa since for no known reason. Recently, only a few adults and larvae of Ae. albopictus has been observed in Sistan and Baluchistan Province, southeastern Iran (23). Knowledge about the behavior of mosqui- toes is very important in the epidemiology of disease transmission and vector control. Data on fluctuations of seasonal abundance of spe- cies can describe their relative risk in the trans- mission of diseases in human populations, and can also help in the planning and implementa- tion of proper control programs (24). Environ- mental changes greatly affect the habitat of mosquito species (25). Meteorological factors also affect the population of mosquitoes by quan- titative and qualitative changes on the larval hab- itats (25, 26). The combination of mosquito be- havior pattern (circadian rhythmicity) with cli- mate factors makes a foundation for determin- ing the timing/month/season of mosquito activ- ities (27, 28). Therefore, determining the sea- sonal prevalence of mosquito fauna in an area is crucial for the development of effective vec- tor control programs and updating of ecologi- cal information related to vectors of diseases in the area (25, 29). There were little published data on the sea- sonal abundance of mosquitoes in Iran (30-32). Up to now, fauna, checklist, physicochemical factors of larval habitats, co-occurrence, asso- ciation, affinity and biodiversity of mosqui- toes have been studied in Mazandaran Province (10, 33-35), however, there is no recent com- prehensive study on the seasonal activity of mos- quitoes in the province. Therefore, this study was conducted to evaluate the monthly dynam- ics of the mosquito populations and their rela- tion to climatic factors in Mazandaran Province. Materials and Methods Study area Mazandaran Province is located on the coast of the Caspian Sea in northern Iran, in coordi- nate latitude 35°47′–36°35′N and longitude 50°34′–54°10′E between the Caspian Sea and the Alborz mountain range. The province is enclosed by Golestan Province in the east, Gui- lan Province in the west and Tehran and Sem- nan Provinces in the south and the Caspian Sea to the North. The diverse nature of the prov- ince included plains, grassland, forests and rain- forest with an area of approximately 23,842 square kilometers and a population of approx- imately 3,073,943. In the study area, the main agricultural products are rice, followed by wheat, http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 209 http://jad.tums.ac.ir Published Online: June 30, 2021 barley, beans, fruits and vegetables. Vast Hyr- canian forests, temperate climate, abundant wet- lands and rice fields in the province, provide enormous adequate habitats for the develop- ment of mosquitoes. Specimen and data collection Mosquitoes were collected from 30 villag- es in 16 counties from Mazandaran Province, in a uniform method, once a month from May to December 2014. In each county, two vil- lages were randomly designated for sampling mosquitoes according to topography of areas. In each village, one fixed habitat was selected for collection of larvae. Larval collection was conducted by a standard 350ml dipper for 15– 20min per natural and artificial breeding sites in fixed habitats. In the larval habitat, 10–30 dips were taken based on the size of the breeding site. Samples were always taken by the same individual in the morning (0800–1200 hours) or afternoon (1400–1800 hours). Adult mosquitoes were collected in eight places (three human and three animal fixed and one human and one animal variable places) be- tween 0500 and 0800 hours in selected villag- es by methods of total catch. Before spraying, all the eaves, windows, doors and other exit points were closed and white cloth sheets were spread on the floor. After Pyrethrum spraying, the room was kept closed for 10min and the knocked-down mosquitoes were then collected from the floor sheet. Pit shelters (90× 150× 150cm W× L× D) were dug in shady places in each village, then small cavities, about 30cm deep were dug horizontally at each side, about 50cm above the bottom of the pit. The col- lections of resting mosquitoes were carried out by aspirator from the walls of the pits between 0500 to 0600 hours. CDC light traps (John W. Hock Company, Gainesville, FL) were set about 1.5–2m above the ground in human and ani- mal dwellings in each village. Light traps were switched on at 1900 and switched off at 0600 hours local time. The mosquitoes were attract- ed to the light at night, and were arrived to fun- nel nets screen using the airflow generated by the fan motor. Human-landing collections (two human baits and one collector) using mouth as- pirators were performed for sampling of Aedes, during the daytime between 0900 and 2100 hours, in each village. Sampling teams were stationed mostly between human and animal sites near appropriate larval habitats. The mos- quitoes were collected at 1h intervals and were placed in paper cups relevant to the same hour. The third and fourth instar larvae were pre- served in lactophenol solution and adults were transferred to cups container with moist cot- ton, labeled and mounted (10). The specimens were identified by direct observation of mor- phological characters using valid taxonomic keys (36). The specimens are deposited at the Mu- seum of Medical Entomology, School of Health, Mazandaran University of Medical Sciences, Sari, Iran. The abbreviations of mosquito gen- era and subgenera are cited by Reinert (37). Meteorological data Monthly meteorological variables containing mean temperature and rainfall were obtained from the synoptic stations of Mazandaran Prov- ince Meteorological Organization in 2014. Abundance Indices “Index of Species Abundance” (ISA) was used to assess the most abundant species of mos- quitoes in the province based on the explana- tions of Robert and Hsi (38) with a minor mod- ification in the formula. ISA is calculated by the following formula: Where ‘a’ is the number of sampling sites that the species not present in it, ‘c’ is the high- est rank of the species in sampling sites plus 1, and ‘Rj’ is the sum of the rankings of each species in all sampling sites, ‘K’ is the num- ber of sampling sites. “Standardized Index of Species Abundance” (SISA) was applied as a standardized formula http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 210 http://jad.tums.ac.ir Published Online: June 30, 2021 for ISA by converting to a scale from zero to one with the following changes: When SISA is closer to 1, it represents the most abundant species (39). Statistical analysis Statistical analysis was performed on data obtained from mosquito population density dur- ing the study to understand whether meteoro- logical variables could be the cause of popula- tion fluctuations of mosquitoes in the study ar- ea. The data were calculated using SPSS Ver. 23. The relationships between monthly popu- lation fluctuations of mosquitoes with mete- orological variables were evaluated by statis- tical test of “Pearson’s correlation coefficient (R)”. Results Overall, 23,750 mosquitoes (7,566 larvae and 16,184 adults) belonging to two subfami- lies, four genera and nineteen species were col- lected and identified throughout the year dur- ing this study. The subfamily Anophelinae was represented by one genus and 7 species, while the subfamily Culicinae was in 3 genera and 12 species. Monthly fluctuations in population dynamic of mosquitoes are displayed in (Fig. 1, Table 1 and 2). The highest total number of larvae (2,383) and adult (4,723) mosquitoes were observed in July and the lowest in May (Fig. 1). Among the larvae, An. maculipennis s.l. and An. pseudopictus were collected dur- ing each month except May and December, respectively, while An. hyrcanus was observed only during June–October. The population den- sity of An. maculipennis s.l. reached its peak in June (n= 238). After June, the population den- sity of the species with an irregular fluctua- tion decreased rapidly and the lowest density was in September (n= 5). The highest popula- tion peak of An. hyrcanus was quite similar to that of An. maculipennis s.l, while it was in July for An. pseudopictus. Culex pipiens and Cx. torrentium was found from May to December. The largest population density of this species was observed in July (n= 1670) and June (n= 287) respectively. Culex tritaeniorhynchus was collected from May to November, with a major peak in July. Culex perexiguus and Cx. mimeti- cus were almost non-active in the first and last seasons of the year and had the maximum number in July. Culiseta annulata and Cs. longi- areolata were almost absent during the warm seasons of the year and their highest activity peaks were recorded in October and Novem- ber, respectively. Anopheles marteri, An. claviger and Cs. morsitans were not found in adequate numbers to draw their monthly activity patterns (Table 1). Among the adults, An. maculipennis s.l. was present almost throughout the year except for December, the population dynamics of this spe- cies starts in May, reaching a major peak in June and then decreases gradually. The popula- tion density of An. hyrcanus begins to increase in May, reaching its greatest peak in June and after that, decreased. The species disappeared from monthly sampling in August, and was ob- served with the lowest population density again in September. Anopheles pseudopictus was rec- orded during June to November, its highest and lowest population peak was in July (n= 914) and November (n= 40), respectively. The den- sity of An. claviger and An. sacharovi was peaked during August (n= 28) and July (n= 87), respectively. Culex pipiens was succes- sively found throughout the monthly sampling period with major peak in July. Since July, the population of the species decreased gradu- ally in August and September, then the activity increases and reaches the smaller peak in De- cember. The population density of Cx. tri- taeniorhynchus among the collected mosquitoes was high in July (n= 1868), it decreased in Au- gust (n= 1288), increased again to the smaller population peak in September (n= 1820), finally decreased progressively to the end of the sea- son. Culex perexiguus, Ae. vexans and Cs. an- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 211 http://jad.tums.ac.ir Published Online: June 30, 2021 nulata had the most activity in June, October and November in the study area. The rest of the species were collected in low numbers, so it was not possible to predict a proper pattern of monthly population fluctuations for them in the study area (Table 2). The highest mean temperature and rainfall was observed during the months of August and October, respectively (Fig. 2). Bivariate Pear- son’s correlation analyses of mosquito popu- lation density with meteorological variables ex- hibited that the population fluctuations of Cx. territans (R= 0.855, P= 0.007), Cs. annulata (R= 0.711, P= 0.0048), Cs. longiareolata (R= 0.826, P= 0.011) and Ae. vexans (R= 0.831, P= 0.011) have significant positive correlation with rainfall in the study area. However, no significant association was observed between other mosquito species and rainfall. The month- ly temperature showed a significant positive correlation with the adult population fluctua- tions of Cx. tritaeniorhynchus (R= 0.766, P= 0.027). As for other species, the temperature was not seen as an important variable in population fluctuations in the study area (Table 3). The in- teraction between mosquito population fluctu- ations with mean monthly temperature and rain- fall is shown in (Fig. 2). The highest number and percentage of mosquitoes were collected by total catch in animal places (n:8051, 49.74%) followed by 16.82% using light trap, 14.87% by total catch in human places, 11.15% using pit shelter and 7.42% by day biting. Among Anophelinae, An. maculipennis s.l. (n: 1555, 86.97%) and An. pseudopictus (n: 680, 52.84%) were the most common in total catch of an- imal places and the least in daily bites and pit shelter, respectively, while An. claviger and An. sacharovi were collected with the highest num- ber and percentage using pit shelter sampling method. Among Culicinae, Cx. tritaeniorhyn- chus is more common than Cx. pipiens using total catch in animal places and light trap, whereas the species was observed with the low- est number and percentage by total catch in hu- man places and pit shelter than Cx. pipiens. Ae- des vexans were collected by all sampling meth- ods, but the species was collected with the highest percentage up to 94.5% (n: 1200) in day biting collections. Further data on other species collected by each trap are shown in Table 4. Based on Table 5, larvae of Cx. pipiens, Cx. torrentium and Cx. tritaeniorhynchus showed values of SISA 1 (ISA= 1), 0.805 (ISA= 3.62) and 0.564 (ISA= 6.87), respectively, whereas it was 0.550 (ISA= 7.06) for An. maculipennis s.l. Among adult mosquitoes, the highest SI- SA (0.822, 0.637) and the lowest ISA (3.31, 5.71) were calculated for An. pseudopictus and An. maculipennis s.l. in total catch performed in animal shelters, respectively. SISA was 0.977 (ISA= 1.25), 0.946 (ISA= 1.594) and 0.933 (ISA= 1.87) in association with Cx. pipiens in total catch performed in human dwellings, pit shelter and total catch performed in animal shel- ters, respectively. It was 0.938 (ISA= 1.68) in pit shelter, 0.938 (ISA= 1.69) in light trap, 0.913 (ISA= 2.125) in total catch carried out in ani- mal places and 0.886 (ISA= 2.25) in total catch performed in human dwellings for Cx. tri- taeniorhynchus. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 212 http://jad.tums.ac.ir Published Online: June 30, 2021 Table 1. Monthly changes in population fluctuations of mosquito larvae in Mazandaran Province, northern Iran May June July August September October November December Total Species No % No % No % No % No % No % No % No % No % An. claviger 0 0 0 0 0 0 15 100 0 0 0 0 0 0 0 0 15 100 An. hyrcanus 0 0 16 40 2 5 9 22.5 9 22.5 4 10 0 0 0 0 40 100 An. maculipennis s.l. 0 0 181 47.14 80 20.83 30 7.81 5 1.30 14 3.64 64 16.67 10 2.60 384 100 An. pseudopictus 0 0 11 7.14 61 39.61 13 8.44 35 22.73 19 12.34 15 9.74 0 0 154 100 An. marteri 0 0 0 0 1 100 0 0 0 0 0 0 0 0 0 0 1 100 Cx. pipiens 75 1.52 1197 24.27 1670 33.87 527 10.69 818 16.59 261 5.29 277 5.61 106 2.15 4931 100 Cx. torrentium 1 0.16 287 46.22 154 24.8 20 3.22 17 2.74 76 12.24 53 8.53 13 2.09 621 100 Cx. tritaeniorhynchus 2 0.27 4 0.53 324 43.32 139 18.58 213 28.48 58 7.75 8 1.07 0 0 748 100 Cx. perexiguus 0 0 0 0 32 65.31 5 10.2 4 8.16 8 16.33 0 0 0 0 49 100 Cx. territans 0 0 0 0 1 1.59 0 0 0 0 44 69.84 18 28.57 0 0 63 100 Cx. mimeticus 1 1.19 0 0 54 64.29 22 26.19 1 1.19 6 7.14 0 0 0 0 84 100 Cx. hortensis 1 20 0 0 0 0 0 0 4 80 0 0 0 0 0 0 5 100 Cs. annulata 15 8.88 27 15.98 0 0 0 0 0 0 60 35.5 67 39.64 0 0 169 100 Cs. longiareolata 0 0 89 29.57 4 1.33 0 0 0 0 119 39.53 80 26.58 9 2.99 301 100 Cs. morsitans 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 100 1 100 Total 95 1.26 1812 23.95 2383 31.5 780 10.31 1106 14.62 669 8.48 582 7.69 139 1.83 7566 100 Table 2. Monthly changes in population fluctuations of adult mosquitoes in Mazandaran Province, northern Iran May June July August September October November December Total Species No % No % No % No % No % No % No % No % No % An. claviger 8 14.55 0 0 2 3.63 28 50.91 14 25.45 3 5.46 0 0 0 0 55 100 An. hyrcanus 14 9.93 95 67.38 21 14.89 0 0 2 1.42 9 6.38 0 0 0 0 141 100 An. maculipennis s.l. 20 1.12 889 49.72 652 36.47 148 8.28 44 2.46 31 1.73 4 0.22 0 0 1788 100 An. pseudopictus 0 0 54 4.19 914 71.02 109 8.47 90 6.99 80 6.22 40 3.11 0 0 127 100 An. marteri 2 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 100 An. sacharovi 0 0 1 0.89 87 77.68 22 19.65 1 0.89 0 0 1 0.89 0 0 112 100 An. superpictus 0 0 0 0 5 55.56 0 0 0 0 0 0 4 44.44 0 0 9 100 Cx. pipiens 426 8.26 918 17.8 1134 21.99 612 11.87 285 5.53 420 8.14 562 10.9 800 15.51 5157 100 Cx. tritaeniorhynchus 0 0 379 6.14 1868 30.26 1288 20.87 1820 29.48 607 9.84 210 3.40 1 0.01 6173 100 Cx. perexiguus 23 25.56 47 52.22 15 16.67 0 0 0 0 5 5.55 0 0 0 0 90 100 Cx. mimeticus 2 16.67 2 16.67 5 41.66 0 0 3 25 0 0 0 0 0 0 12 100 Cx. hortensis 0 0 0 0 1 100 0 0 0 0 0 0 0 0 0 0 1 100 Ae. vexans 5 0.39 0 0 11 0.87 9 0.71 4 0.31 1202 94.65 39 3.07 0 0 1270 100 Ae. caspius 7 18.42 21 55.26 8 21.05 0 0 0 0 1 2.63 0 0 1 2.63 38 100 Cs. annulata 1 2.04 0 0 0 0 2 4.08 0 0 2 4.08 44 89.8 0 0 49 100 Total 508 3.14 2406 14.87 4723 29.18 2218 13.70 2263 13.98 2360 14.58 904 5.59 802 4.96 16184 100 http://jad.tums.ac.ir Published Online: June 30, 2021 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 213 http://jad.tums.ac.ir Published Online: June 30, 2021 Table 3. Pearson’s correlation coefficient (R) of mosquito population density with meteorological variables in Ma- zandaran Province, northern Iran Species Temperature Rainfall Larvae R P R P An. claviger 0.394 0.335 -0.374 0.361 An. hyrcanus 0.630 0.094 -0.143 0.735 An. maculipennis s.l. 0.236 0.574 0.001 0.998 An. pseudopictus 0.548 0.159 -0.263 0.529 Cx. pipiens 0.666 0.710 -0.364 0.379 Cx. torrentium 0.314 0.449 0.136 0.747 Cx. tritaeniorhynchus 0.667 0.071 -0.478 0.231 Cx. perexiguus 0.441 0.274 -0.179 0.672 Cx. territans -0.260 0.534 0.855 0.007 Cx. mimeticus 0.496 0.211 -0.350 0.395 Cs. annulata -0.400 0.326 0.711 0.048 Cs. longiareolata -0.238 0.571 0.826 0.011 Adults An. claviger 0.570 0.140 -0.546 0.162 An. hyrcanus 0.295 0.478 0.051 0.904 An. maculipennis s.l. 0.489 0.218 -0.166 0.694 An. pseudopictus 0.426 0.293 -0.289 0.488 An. sacharovi 0.444 0.270 -0.379 0.354 Cx. pipiens 0.060 0.887 -0.115 0.786 Cx. tritaeniorhynchus 0.766 0.027 -0.475 0.234 Cx. perexiguus 0.292 0.483 -0.48 0.911 Cx. mimeticus 0.584 0.128 -0.523 0.183 Ae. vexans -0.086 0.840 0.831 0.011 Ae. caspius 0.298 0.474 -0.066 0.877 Cs. annulata -0.475 0.235 0.140 0.741 Fig. 1. The total number of mosquitoes collected by month in Mazandaran Province, northern Iran http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 214 http://jad.tums.ac.ir Published Online: June 30, 2021 Table 4. Number and percentage of adult mosquitoes collected based on sampling method Sampling method Total catch in human places Total catch in animal places Pit shelter Light trap Daily biting Total No % No % No % No % No % No % An. claviger 11 20 20 36.36 24 43.64 0 0 0 0 55 100 An. hyrcanus 7 4.97 89 63.12 2 1.42 44 31.21 0 0 141 100 An. maculipennis s.l. 91 5.09 1555 86.97 41 2.29 101 5.65 0 0 1788 100 An. marteri 0 0 2 100 0 0 0 0 0 0 2 100 An. pseudopictus 88 6.84 680 52.84 81 6.30 438 34.03 0 0 1287 100 An. sacharovi 0 0 2 1.78 108 96.44 2 1.78 0 0 112 100 An. superpictus 0 0 9 100 0 0 0 0 0 0 9 100 Cx. pipiens 1437 27.87 2225 43.14 870 16.87 625 12.12 0 0 5157 100 Cx. tritaeniorhynchus 700 11.34 3371 54.60 621 10.06 1481 24 0 0 6173 100 Cx. perexiguus 27 30 25 27.77 18 20 20 22.22 0 0 90 100 Cx. mimeticus 1 8.33 10 83.33 0 0 1 8.33 0 0 12 100 Cx. hortensis 0 0 1 100 0 0 0 0 0 0 1 100 Ae. vexans 43 3.38 16 1.25 5 0.39 6 0.47 1200 9.54 1270 100 Ae. caspius 0 0 13 34.21 24 63.15 0 0 1 2.63 38 100 Cs. annulata 2 4.08 33 67.34 10 20.40 4 8.16 0 0 49 100 Total 2407 14.87 8051 49.74 1804 43.64 2722 16.82 1201 7.42 16184 Table 5. Abundance indices (ISA/SISA)* of the most abundant species (lower ISA values and SISA closer to 1) of mosquitoes collected based on sampling method in Mazandaran Province, northern Iran Larvae Adult Total catch in human places Total catch in animal places Pit shelter Light trap Daily biting Species SISA ISA Species SISA ISA SISA ISA SISA ISA SISA ISA SISA ISA An. claviger 0.000 24.06 An. claviger 0.000 20.97 0.000 21.22 0.000 20.88 0.000 21.3 0.000 17.1 An. hyrcanus 0.000 14.5 An. hyrcanus 0.000 18.78 0.000 14.09 0.000 20 0.000 16.4 0.000 17.1 An. maculipennis s.l. 0.550 7.06 An. maculipennis s.l. 0.369 7.93 0.637 5.719 0.000 12.78 0.000 16 0.000 17.1 An. pseudopictus 0.273 10.81 An. pseudopictus 0.454 7 0.822 3.313 0.46 6.938 0.668 4.66 0.000 17.1 An. marteri 0.000 24.5 An. marteri 0.000 22.19 0.000 23.97 0.000 20.75 0.000 22 0.000 17.1 Cx. pipiens 1 1 An. sacharovi 0.000 22.19 0.000 23.94 0.000 20.69 0.000 19.8 0.000 17.1 Cx. torrentium 0.805 3.62 An. superpictus 0.000 22.19 0.000 22.44 0.000 22.06 0.000 22 0.000 17.1 Cx. tritaeniorhynchus 0.564 6.87 Cx. pipiens 0.977 1.25 0.933 1.87 0.946 1.59 0.696 4.34 0.000 17.1 Cx. perexiguus 0.000 17 Cx. tritaeniorhynchus 0.886 2.25 0.913 2.12 0.938 1.68 0.938 1.69 0.000 17.1 Cx. territans 0.000 19.19 Cx. perexiguus 0.000 17.53 0.000 18.03 0.000 20.78 0.000 20.6 0.000 17.1 Cx. mimeticus 0.037 14 Cx. mimeticus 0.000 21.13 0.000 19.19 0.000 22.06 0.000 20.9 0.000 17.1 Cx. hortensis 0.000 21.69 Cx. hortensis 0.000 22.19 0.000 23.94 0.000 22.06 0.000 22 0.000 17.1 Cs. annulata 0.000 15.12 Ae. vexans 0.000 19.75 0.000 22.63 0.000 19.78 0.000 19.8 0.1 9 Cs. longiareolata 0.152 12.44 Ae. caspius 0.000 22.19 0.000 23.69 0.000 19.66 0.000 22 0.000 16.1 Cs. morsitans 0.000 22.69 Cs. annulata 0.000 19.97 0.000 16.34 0.000 16.47 0.000 18.6 0.000 17.1 http://jad.tums.ac.ir Published Online: June 30, 2021 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 215 http://jad.tums.ac.ir Published Online: June 30, 2021 *ISA: Index of Species Abundance; SISA: Standard Index of Species Abundance Fig. 2. Relation between population fluctuations of larvae/adult mosquitoes and meteorological variables in Mazandaran Province, northern Iran http://jad.tums.ac.ir Published Online: June 30, 2021 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 216 http://jad.tums.ac.ir Published Online: June 30, 2021 Discussion Demonstration of the monthly changes in the population abundance of mosquito species, along with meteorological parameters is cru- cial in understanding the biology of potential vector species for efficient mosquito control strategies. This is the first comprehensive study on this subject in Mazandaran Province, north- ern Iran using the ISA/SISA indices in order to identify the most abundant species. Vector abundance is a key determining factor that is often used as a risk indicator of vector-borne diseases. It is quantitatively more important than vector competence (although competence is a transmission requirement). It could explain the importance of abundant species in an area and why some vectors could contribute to epidemics without considering the competence for trans- mission (40-41). Culex pipiens is the most abun- dant species both in the larval and adult stages based on indices of SISA/ ISA in the present study. It was noted to be a common species in other studies in different parts of Iran (35, 42- 45), however, it should be mentioned that none of these researches reported abundance of spe- cies based on the SISA/ISA indices. Although Cx. pipiens was collected most frequently by total catch in animal places, it was the predom- inant species (based on indices of SISA/ISA) in total catch in human places and pit shelter compared to other species, respectively. It in- dicates that the species tends to be attracted to humans, animals and also shows some extent of exophily in the study area. It is possible that humans come into contact with pathogens this species may carry. Such studies could estab- lish baseline data for public health interventions in control programs, therefore, an assessment of different trapping methods is also required to adjust the design of future entomological and pathogen surveillance efforts (46). The monthly activity of Cx. pipiens (larvae and adult) be- gins in mid-spring after diapause termination and reaches its largest peak in July and then declines with irregular fluctuations until the species disappears. It seems that the spring rains are cause for the population of the species to peak in July, while no significant association was observed between Cx. pipiens and mete- orological factors, probably high compatibil- ity and access to diverse breeding places could help increasing population density of the species in early warm season. In contrast, Cx. pipiens was active from May to November and June to September (29, 47). The population density of this species increased in July (47) and May to August (48), reaching its largest peak in August (47-48). Culex pipiens showed an in- crease in population density from May to July in north-eastern Croatia, then sharp decline oc- curred toward the end of the season (49). In northern Italy, the species had maximum ac- tivity peak in July (50), that is almost consistent with the results of the present study. There is not much data about the seasonal activity of culicine mosquitoes, especially in adult stage, in Iran (30). The highest activity peak of Cx. pipiens was documented in mid-July in Gui- lan Province. There is no significant correla- tion between species population density and the meteorological data (30), which is in accord- ance with our research. Variations in the seasonal patterns of the species in this study and find- ings in other regions can probably be due to the differences in the topography and climates. Culex pipiens with its ornithophilic and op- portunistic feeding behavior that bites both hu- mans and animals, can be as bridge vector be- tween birds and humans so is believed to be the principal vector of West Nile Virus (51). It also shows an important role in transmis- sion of several human pathogens including St. Louis encephalitis virus (SLEV), filarial worms as well as wildlife pathogens such as avian malaria (52). The existence of swamps for mi- grating birds and their active presence through- out the autumn and winter in Mazandaran Prov- ince and detection of virus in the species in Guilan Province (53) can cause a concern for http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 217 http://jad.tums.ac.ir Published Online: June 30, 2021 entry and the spread of the virus in the region. Culex tritaeniorhynchus, the second most abun- dant species based on indices of SISA/ISA in the study area, was first collected in May (lar- val stage) and June (adult stage), increased sharply in July, becoming the most abundant species, and displayed a rapid decline in De- cember. The abundant species shifted from Cx. tritaeniorhynchus to Cx. pipiens in November, probably due to high adaptability of Cx. pipiens to the environmental conditions in the area. In contrast, this species showed the highest peak of activity in August in Belek Region of Tur- key (29). While the peak of activity was ob- served in July in Guilan Province (30), which is in agreement with our study. Culex tritaeniorhyn- chus is caught with the highest number by to- tal catch in animal places which reflects the zoophilic tendencies of the species in the study area. Based on the results of a study in India, it is considered to be predominantly exophilic and normally zoophilic (54). It was recorded to be the most abundant species in numerous studies in Iran, without reporting any SISA/ ISA indices (33-34, 44-55). Culex tritaeniorhyn- chus is the primary vector of Japanese enceph- alitis (JE) in southern Asia. It has also been observed infected with dengue fever, Rift Valley fever, Sindbis, Getah and Tembusu viruses, and the filariae of both Brugia malayi and Wu- chereria bancrofti, in many areas of eastern and southeastern Asia (55-56). The species is a principle vector of Rift Valley fever in Sau- di Arabia (57), WNV in Asia (9) and a possi- ble vector of Japanese encephalitis in Iraq (58), which indicates a high risk species for human health. Among the Anopheles, An. maculipennis s.l. is numerically the most abundant species in the present study, mostly sampled by total catch method in animal places, displayed to be predominantly zoophilic. This is consistent with other studies, but there is no evidence of SISA/ISA indices to express the most abun- dant species in these studies (43, 59-61). What is interesting is that, despite having the high- est numerical abundance of species, An. pseu- dopictus was calculated as the most abundant species in all sampling methods (except daily bites) based on ISA/SISA indices. In addition, An. sacharovi also had the highest numerical abundance in the pit shelter, but it was not con- sidered as the most abundant species based on the indices of SISA/ISA. Moreover, An. sacha- rovi was the most abundant species in the pit shelter, but only with regards to its high nu- merical abundance. Therefore, this shows the importance of species distribution at different sites and computational value of the ISA/SISA indices. Anopheles maculipennis s.l. is known to be the most important malaria vector in northern and western parts and the central plat- eau of Iran (62). Moreover, there is a belief that this species can play a vector role in WNV trans- mission in various countries of the old world (63, 64). Recently, the myxoma virus genome was detected in wild caught An. maculipennis that fed on wild rabbits by polymerase chain reaction (PCR) in southern England, UK. Batai virus (BATV) and Anopheles associated C vi- rus (AACV) was also identified and isolated from An. maculipennis complex in entomolog- ical surveys in Germany, Italy and France (65). With regards to the malaria historical records and WNV in the northern parts of Iran (53, 66), understanding the population dynamics and the maximum monthly peaks of the species is im- portant in controlling the diseases in the study area. The highest monthly activity peak of An. maculipennis (larvae and adults) is in June. In contrary to our results, An. maculipennis s.l. showed the highest peak of activity in the mid- July in Guilan (30) and July–August in Kalaleh County of Golestan Province (31), northern Iran. In neighboring Turkey, the species peaked in August and July–August (67-68). These dif- ferences in population patterns could possibly be due to regional ecological differences. On the other hand, some researchers believe that these discrepancies might also be due to the low collection effort involved in the studies (27) or sampling regimes (29-30, 50, 67-68). Climate http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 218 http://jad.tums.ac.ir Published Online: June 30, 2021 change and the environment affect the abun- dance and distribution of vectors and their in- termediate hosts (69). In the present study, monthly population fluctuations of Cs. annu- lata, Cs. longiareolata and Ae. vexans are cor- related with rainfall. Although these species showed irregular monthly population fluctua- tions, they were more active in spring and au- tumn. Culiseta annulata had a major peak in November (29), which is in accordance with our research. On the other hand, Cs. longiare- olata was active in July and September (67), whereas in the present study, the species had the largest peak in October, which is quite similar to that of Ae. vexans. The largest peak of Ae. vexans was reported in June (67) and August (68). There are evidence that Ae. vexans and Cs. annulata are important vectors of WNV (70-71). Among the tribe Aedini, Ae. vexans is recorded to be the most abundant species based on ISA/SISA indices. It was caught in all traps and is the most abundant collected species in day biting method compared with other species, and shows anthropophilic tenden- cies in the study areas. This species demon- strated high prevalence and collected by day biting method in other studies performed in different parts of Iran (59, 72-74), this may be a reflection of the fact that this method has a high level of efficiency for collecting this spe- cies. However, to observe ethical values, this collection methods cannot be used freely and with extended time, and should in fact be re- stricted or modified in compliance with ethi- cal standards (75). The species is known to be the main vector of Tahyna virus in central Eu- rope (76), potential vector of the dog heart- worm Dirofilaria immitis in Europe (77) and principal vectors of Rift Valley fever in Saudi Arabia (57). Recently, Zika virus is revealed in the salivary glands of the field-caught Ae. vexans (78). It is a flood water mosquito, so, their population abundance depends on the water dynamics of temporary pools (27). Rain- fall as an important climatic factor may have a range of different correlation with the popula- tion of the species, from positive, negative and/ or ineffective and sometimes with a lag phase (79), a significant positive correlation between its monthly activity and rainfall was observed in our study. The current study was not designed and aimed to address the analysis of lag time between climate factors and population densi- ty of the species, however, it seems that there is probably a lag time of at least 15 days after rain at the beginning of the rainy season be- fore the Ae. vexans population jump start. In concordant with our findings, there is a lag time of at least 15 days between the peak of rainfall and abundance of the species (80-82). Moreover, other studies have revealed a cor- relation between rainfall and the abundance of the species with a 10 days lag in the early rainy season and 20 days after the end of the rainy season (83). In the study of Diallo et al. (82) rainfall patterns displayed that heavy rains be- tween August and September 2005 had nega- tively impacted on the population density of Ae. vexans, whereas in 2006, population peak was observed following the rainfall peak. These findings show the complexity of the relationship between climate factors and population density of mosquitoes. Therefore, it is recommended to measure the seasonal activities of species over multiple years, to show a better understanding of the correlation between population frequen- cy and climatic factors, and the impact of other variables. The highest number and population density of Ae. vexans was found during early autumn, especially in October. Vector-borne dis- eases show seasonal patterns with inter and in- tra-annual variability, which are partly described by climate and environmental factors (80). There- fore, these results are important for health au- thorities in controlling mosquitoes as well as in the tourism industry for nuisance control, but more importantly, it provides a detailed esti- mate of the timing of risk for human populations. Conclusion Based on ISA/SISA indices, July in which http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2021, 15(2): 207–224 SH Nikookar et al.: Population Fluctuations and … 219 http://jad.tums.ac.ir Published Online: June 30, 2021 Cx. pipiens and Cx. tritaeniorhynchus have the highest population peak, is the most crucial time for efficient mosquito control program in the area study. Acknowledgements We would like to thank the staff of Ma- zandaran Provincial Health Deputy for their direct help in the field sampling. Also we ex- press our appreciation to the people of the vil- lages for their kind assistance with the sam- pling teams during the study. The study was supported by the Deputy for Research and Tech- nology of Mazandaran University of Medical Sciences, Mazandaran, Iran by grant No. 93– 1017. The authors declare that there is no con- flict of interest. References 1. Rueda LM (2008) Global diversity of mos- quitoes (Insecta: Diptera: Culicidae) in freshwater. 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