J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 376 http://jad.tums.ac.ir Published Online: December 31, 2020 Original Article Employing Different Traps for Collection of Mosquitoes and Detection of Dengue, Chikungunya and Zika Vector, Aedes albopictus, in Borderline of Iran and Pakistan Jalil Nejati1; Morteza Zaim2; *Hassan Vatandoost2,3; *Seyed Hassan Moosa-Kazemi2; Rubén Bueno-Marí4; Shahyad Azari-Hamidian5; Mohammad Mehdi Sedaghat2; Ahmad Ali Hanafi- Bojd2,3; Mohammad Reza Yaghoobi-Ershadi2; Hassan Okati-Aliabad1; Francisco Collantes6; Ary A. Hoffmann7 1Health Promotion Research Center, Zahedan University of Medical Sciences, Zahedan, Iran 2Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Department of Environmental Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran 4Departamento de Investigación y Desarrollo (I+D), Laboratorios Lokímica, Valencia, Spain 5Department of Health Education, Research Center of Health and Environment, School of Health, Guilan University of Medical Sciences, Rasht, Iran 6Department of Zoology and Physical Anthropology, University of Murcia, Murcia, Spain 7Bio21 Institute, Pest and Environmental Adaptation Group, School of BioSciences, University of Melbourne, Victoria, Australia *Corresponding authors: Dr Hassan Vatandoost, E-mail: hvatandoost1@yahoo.com, Dr Seyed Hassan Moosa- Kazemi, E-mail: moosakazemi@tums.ac.ir (Received 25 May 2020; accepted 06 Dec 2020) Abstract Background: Southeastern Iran has been established as an area with the potential to harbor Asian tiger mosquito popu- lations. In 2013, a few numbers of Aedes albopictus were detected in three sampling sites of this region. This field study was aimed to evaluate the efficacy of various traps on monitoring mosquitoes and status of this dengue vector, in five urban and 15 suburban/rural areas. Methods: For this purpose, four adult mosquito traps (BG-sentinel 2, bednet, Malaise, and resting box trap) were used and their efficacy compared. In addition, large numbers of CDC ovitraps were employed, within 12 months. Results: A total of 4878 adult samples including 22 species covering five genera were collected and identified from traps. It was not revealed any collection of Ae. albopictus. Statistical analysis showed no significant difference in mete- orological variables between the two periods, the previous report and the current study. There were significant differ- ences in the total number of mosquitoes collected by various traps in the region across different months. Conclusion: The resulting data collected here on the efficiency of the various trap types can be useful for monitoring the densities of mosquito populations, which is an important component of a vector surveillance system. While the pres- ence of Ae. albopictus was determined in this potential risk area, there is no evidence for its establishment and further monitoring needs to be carried out. Keywords: Stegomyia albopicta; Ovitrap; Sistan and Baluchistan Introduction Like many countries around the globe, the dengue vector, Aedes albopictus, has been re- cently detected in Iran, with the first report of this species being in 2016 (1). Considering a previous report about the presence and estab- lishment of this species in the eastern neigh- bor of Iran, Pakistan, this was not unexpected (2-4). Despite its short flight length, Ae. al- bopictus is considered an invasive species with a rapid potential expansion. It has been Copyright © 2020 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:hvatandoost1@yahoo.com mailto:moosakazemi@tums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 377 http://jad.tums.ac.ir Published Online: December 31, 2020 able to spread and establish in various areas from Southeast Asia to America, Europe, Af- rica and Australian regions (5-7). Due to cli- matic changes, global warming, and increased international traveling, arboviral diseases and their vectors have been expanded from en- demic areas (8). The first Iranian dengue fe- ver case with travel background to Southeast Asia was reported in 2008 (9). The health sys- tem expressed its growing concern after DENV- IgG detection in blood donors in southeast of the country, an Oriental ecozone situated in the transit route between East Asia and other countries (10, 11). This area has also been es- timated as potentially harboring Ae. albopic- tus (12). This hypothesis was verified by the detection of Ae. albopictus, although only five larvae and seven adults were collected (1). Therefore, it is crucial to ascertain the estab- lishment status of this species based on eco- logical studies and using various sampling methods (13). Indeed, the efficiency of differ- ent mosquito traps needs to be investigated across a region to establish an effective vector surveillance system (14). Using a range of trap types, this study tests whether Ae. al- bopictus is established in southeastern Iran and explores the presence of other diurnally active mosquitoes in this potential risk area. Materials and Methods Study area This study was carried out in Sistan and Baluchestan, the largest province in Iran (181, 785km²). It includes 19 cities, 37 towns, and 9716 villages. It has a long shared border with Pakistan and Afghanistan in the east and the Arabian Sea in the south. Aridity, dust storms, and especially the so-called ‘Wind of 120 Days’ constitute its general climate. Nevertheless, pe- riodic monsoon systems contribute to a di- verse climate in the region, especially in southern coastal areas where there are sum- mer rainfalls (11). The maximum average rain- fall within 5 years (2011–2015), has been 145.6±89.1mm in the south and 87.9±45.5mm in the northern part of this province. While the region does not possess numerous water bodies (12), cement water tanks (ponds,) as usual water sources especially in rural areas, could be considered mosquitoes breeding plac- es. The humidity in this region is mainly due to the Bengal Gulf streams, which come to Iran only in the summer (15). Despite the growing of date palm gardens, small farms, and even paddy fields in this province (11), the normal- ized difference vegetation index (NDVI) shows a thin vegetation cover (12). Based on biogeo- graphical considerations, this province has a unique climate including the Palaearctic and Indo-Malaya Realms (11). This part of the coun- try still struggles with malaria and other vec- tor-borne diseases, resulting in the establish- ment of a substantial vector surveillance sys- tem in the area (16, 17) (Fig. 1). Traps description The CO2-baited bednet tarp prepared for this study was 2×2×1.2m in size, with 156 threads per inch mesh size and constructed from polystyrene fabrics. CO2 gas was re- leased using a pressure reducing regulator with about 0.5 l/min applied in pulses (20s on/ 40s off) (Fig. 2a). The BG-sentinnel 2 trap con- sisted of a black collapsible fabric container with 40cm high and 36cm in diameter. It has a white gauze lid with a black catch pipe that is opened in case of air suction by an electrical fan. The created airflow, released a BG lure (18) (Fig. 2b). Figure 2c shows the Malaise trap used (height 190–110cm, length 165cm, width 115cm) with a pyramid of white net on a black framework, fabricated from polysty- rene. Two sachets of the new BG-sweet scent were used as a mosquito attractant in this trap. The sachets were hung on the inner side of the black framework. Two types of resting box trap (35×35×35cm) were used, depending on the color of the outer walls (black or black- white banded). The internal walls were cov- ered with black cotton fabric. On the front side, there was a 35×15cm entry near the top, while the rest of the front was covered by a http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 378 http://jad.tums.ac.ir Published Online: December 31, 2020 black wooden\cotton wall. A sugar-ferment- ing yeast solution was applied to attract mos- quitoes. The solution was made up of a mix- ture of dry yeast (7g), sugar (100g) and water (1L) (19) in 1.5L bottles\jars placed in traps or out of them with a connector plastic tube (Fig. 2d). Mosquitoes in this trap (as well as the bednet and Malaise traps) were collected with an oral tube aspirator. The CDC ovitrap is a standardized device for mosquito egg- laying. It involves a black glass jar which is 12.7cm high and 7.6cm in diameter at the top (Fig. 2e). As an oviposition substrate, a wood- en paddle (2.7cm long and 1.9cm wide) was placed in the jar, which was filled with water (20). In our study, the glass jar was replaced by a black plastic pot (19×14cm) due to its lower weight and breaking probability. Field sampling procedure and statistical comparison This descriptive–analytic study was con- ducted from July 2016 to June 2017. In total, five urban (Zahedan, Rask, Nikshahr, Chaba- har and Konarak) 12 rural, and three suburban areas were explored for mosquitoes. In our study, suburb means peri-urban which in- cludes outer residential areas of a city. The sampling points were chosen based on a pre- vious study which had resulted in the Ae. al- bopictus collection. The larvae of this species had been detected in Rask (26.28471 °N, 61.40040 °E; elevation 421 meter above sea level [MASL]) and adult samples were col- lected from Paroomi (25.44267°N, 60.90731°E; elevation 44 MASL) and Vash- name-dori (25.45919°N, 60.83179°E; eleva- tion 9 MASL) (1). Besides, the sampling points were exploited from a modeling study conducted in this area covering various sub- climates and topographies. Adult sampling was undertaken at 10 points; three were fixed (Rask, Paroomi, Vashname-dori) and seven were varied within an area. The first fixed sampling point was urban, and the other two points were classified as rural areas. Sampling started before noon until half an hour after dusk. Adult mosquito traps were installed 20– 50m apart. During adult sampling, meteoro- logical variables including temperature, hu- midity and wind speed were recorded every two hours (Fig. 3b). The CO2-baited bednet, Malaise and Resting box tarps were checked every 15 minutes for five minutes. However, it was done once only at the end of the sam- pling day for the BG-sentinel 2 trap. Due to air suction by the electrical fan, the collected mosquitoes could not escape. Like other traps, the BG-sentinel 2 trap is installed outdoors. Trap placement was conducted according to the instruction manual provided by Biogents AG. It was protected from wind, rainfall, and direct sunlight, yet visible to mosquitoes. Be- sides, the trap was positioned close to the mosquito breeding sites. The collected adult specimens were identified based on an Iranian mosquito key (21). In addition, 505 ovitraps were installed at 31 points (Fig. 3a), including cities, villages, customs areas, and seaports. The paddles were checked every two weeks and suspected cases were moved to the insectary for laboratory assessments. Finally, the obtained data, collected specimens, month, average me- teorological variables and trap types were an- alyzed statistically. Contingency analysis was used to assess the co-occurrence of a species at a location/time point and computed phi from contingency tables as a measure of the strength of species’ association of species between dif- ferent trap types. To this end, generalized lin- ear models (GLMs), were ran, and the data were analyzed as a dichotomous variable in IBM SPSS Statistics 24. Results Adult sampling results A total of 4878 adults were collected from various traps, in which 22 species of five gen- era (including three of the genus Aedes, nine Culex, eight Anopheles, one Culiseta, and one Uranotaenia) were identified. Overall, Ae. caballus (35.5%) and Culex quinquefasciatus (23.82%) were the dominant species, followed http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 379 http://jad.tums.ac.ir Published Online: December 31, 2020 by Cx. sitiens (17.6%), Cx. tritaeniorhynchus (10.9%) and Ae. caspius (4.2%) (Table 1). When pooled across species, a clear mosquito col- lection peak was detected in February, which coincided with a period of low wind speed, low temperature, and high humidity (Fig. 4). Of the 11 meteorological stations scattered throughout the study area, three points were selected. Based on the previous report, Ae. al- bopictus was collected from three sampling sites. The adults had been trapped in Chaba- har County in 2013, and the larvae were col- lected in Nikshahr and Rask Counties in 2009. Thus, meteorological data including the mean temperature as well as mean relative humidity along with precipitation related to these three counties were obtained from the data center of the provincial meteorological organization. Fig- ure 5 shows the monthly comparison of these variables. In December 2013, the maximum rainfall (38.5mm) was recorded in Chabahar station, followed by February of the same year (20.4mm). In 2017, the maximum rainfall oc- curred in February (68.9mm). The Mann-Whit- ney test revealed no significant difference con- cerning these three variables between two years (12 months); 2013 and 2016–2017 (P> 0.05), in three larvae and adult sampling sites (Chaba- har, Nikshahr, and Rask). Most mosquitoes were collected by CO2-baited bednet traps (65.13%), and with a substantial percentage were caught by Malaise traps (34.09%). The Resting-box traps and BG traps only collected 0.51% and 0.27% of the mosquitoes respec- tively. The percentage of species collected by Malaise and bednet traps tended to be highly correlated (Table 1), whereas it varied in the case of other adult trap types (for instance, BG traps captured a high percentage of Cx. si- tiens). Contingency tables (Table 2) indicated that the two trap types placed at the same lo- cation tended to collect the same species. This was also reflected in the high phi scores asso- ciated with these comparisons. When pooled across trap types, the presence/absence of spe- cies was only associated with Cx. quinquefas- ciatus and Cx. tritaeniorhynchus (X2= 15.90, df= 1, P< 0.001, phi= 0.375) as well as Cx. sitiens and Ae. caspius (X2= 36.04, df= 1, P< 0.001, phi= 0.565). The data for these four most common species were statistically ana-lyzed. Another common species, Ae. caballus, was only de-tected in rural areas in one period and it was not analyzed further. Aedes ca-ballus was only collected in February by both Ma- laise and bednet trap types. The other species were collected throughout the year (Fig. 6). Bednet and Malaise trap data were combined to evaluate the effects of the three regions (urban, suburban, and rural) along with other environmental variables on the occurrence of the four common species. For Ae. caspius, GLM indicated the significant effect of region (Wald X2= 9.846, df= 2, P= 0.007), such that this mosquito was more common in urban ar- eas (Fig. 7). Moreover, wind was found to ex- ert a marginally non-significant effect (Wald X2= 3.458, df= 1, P= 0.063), such that lower wind speed was associated with a higher probability of positive traps (B= 1.221, 95% CI 0.066, 2.509). For Cx. quinquefasciatus, GLM only confirmed the significant impact of wind speed (Wald X2= 5.153, df= 1, P= 0.023), such that lower wind speed again was related to positive traps (B= 1.699, 95%, CI 0.232, 3.167). Regarding Cx. tritaeniorhynchus, the results of GLM exhibited the significant effect of location, as reflected by the impact of latitude (Wald X2= 6.708, df= 1, P= 0.010) and longitude (Wald X2= 6.773, df= 1, P= 0.009). Additionally, this species was signifi- cantly affected by wind speed (Wald X2= 5.402, df= 1, P= 0.020). Finally, none of the parameters had a significant role in the occur- rence of Cx. sitiens. Ovitrap results No Aedes eggs and larvae were detected on ovitraps’ paddles and waters respectively. Two species, Cx. tritaeniorhynchus (n= 11) and Anopheles stephensi (n= 4), were collect- ed from water of ovitraps at two points locat- ed in Sarbaz County (26.14118 °N, 61.45337 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 380 http://jad.tums.ac.ir Published Online: December 31, 2020 °E, elevation 329 MASL) and (26.15303°N, 61.44254 °E, elevation 360 MASL). Table 1. The collected mosquito species by trap types, southeastern Iran, Sistan and Baluchestan Province, 2016–2017 Species Co2-baited Bednet Malaise BG Resting Box Total n % n % n % n % n % Aedes caspius 94 3.0 107 6.4 3 12 1 7.7 205 4.20 Ae. caballus 1031 32.5 698 42.0 4 16 1 7.7 1734 35.55 Ae. vexans 2 0.1 1 0.1 0 0 0 0 3 0.06 Culex quinquefasciatus 798 25.1 354 21.3 5 20 5 38.5 1162 23.82 Cx. tritaeniorhynchus 335 10.5 191 11.5 3 12 2 15.4 531 10.89 Cx. pipiens 87 2.7 19 1.1 0 0 3 23 109 2.23 Cx. sitiens 602 18.8 247 14.8 10 40 0 0 859 17.61 Cx. prexigus 38 1.2 10 0.6 0 0 0 0 48 0.98 Cx. bitaniorhynchus 5 0.2 0 0.0 0 0 0 0 5 0.10 Cx. theileri 5 0.2 1 0.1 0 0 0 0 6 0.12 Cx. pseudovishnui 15 0.5 2 0.1 0 0 1 7.7 18 0.37 Cx. siniticus 41 1.3 21 1.3 0 0 0 0 62 1.27 Anopheles stephensi 61 1.9 9 0.5 0 0 0 0 70 1.44 An. culicifacies s.l. 16 0.5 0 0.0 0 0 0 0 16 0.33 An. superpictus s.l. 10 0.3 0 0.0 0 0 0 0 10 0.21 An. turkhudi 1 0.0 0 0.0 0 0 0 0 1 0.02 An. subpictus s.l. 5 0.2 0 0.0 0 0 0 0 5 0.10 An. fluviatilis s.l. 2 0.1 0 0.0 0 0 0 0 2 0.04 An. dthali 1 0.0 0 0.0 0 0 0 0 1 0.02 An. moghulensis 1 0.0 0 0.0 0 0 0 0 1 0.02 Urantaenia unguiculata 2 0.1 0 0.0 0 0 0 0 2 0.04 Culisets longiareolata 25 0.8 3 0.2 0 0 0 0 28 0.57 Total 3177 100 1663 100 25 100 13 100 4878 100 Fig. 1. Location of the study area, Sistan and Baluchestan Province, southeastern Iran, surveyed during 2016–2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 381 http://jad.tums.ac.ir Published Online: December 31, 2020 Table 2. Co-occurrence of mosquitoes from the same species in the two trap types, Sistan and Baluchestan Province, 2016–2017 Species Present in both B and M Absent in both B and M Only in B Only in M X2 (df= 1), P Phi Aedes caspius 26 83 2 2 92.56, <0.001 0.905 Culex quinquefasciatus 58 19 35 1 21.71, <0.001 0.438 Cx. tritaeniorhynchus 29 60 21 3 30.92, <0.001 0.587 Cx. sitiens 17 91 5 0 82.77, <0.001 0.856 B: CO2-baited bednet Trap, M: Malaise Trap Fig. 2. Traps used for mosquito collection, CO2-baited bednet tarp (a), Malaise trap (b), 450 resting box (c), BG (d), ovitrap (e), southeastern Iran, Sistan and Baluchestan Province, 2016–2017 Fig. 3. Mosquito sampling sites; Adults (a), Ovitraps (b), southeastern Iran, Sistan and Baluchestan Province, 2016–2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 382 http://jad.tums.ac.ir Published Online: December 31, 2020 Fig. 4. The collected mosquitoes and meteorological variables by month, southeastern Iran, Sistan and Baluchestan Province, 2016–2017 Fig. 5. Monthly comparison of the meteorological variables between two years, 2013 and July 2016–June 2017 in three sampling sites: (a) Rask, (b) Nikshahr, (c) Chabahar, southeastern Iran, Sistan and Baluchestan Province, 2016–2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 383 http://jad.tums.ac.ir Published Online: December 31, 2020 Fig. 6. Monthly number of mosquitoes from traps as an estimate of population density for (a) Aedes caspius, (b) Ae. caballus, (c) Culex quinquefasciatus, (d) Cx. tritaeniorhinchus and (e) Cx. sitiens. Note log scale apart from (b), Sistan and Baluchestan Province, 2016–2017 Fig. 7. Box plots for ln density of five common species by two traps in different areas. (Ae. cab) Aedes caballus, (Ae. cas) Ae. caspius, (Cx. qui) Culex quinquefasciatus, (Cx. sit) Cx. sitiens, (Cx. tri) Cx. tritaeniorhynchus are plotted. Ln counts from (R) rural, (SU) suburb, and (U) urban regions are plotted separately. Traps were (B) bednet or (M) Malaise. Asterisks indicate outliers, Sistan and Baluchestan Province, 2016–2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 384 http://jad.tums.ac.ir Published Online: December 31, 2020 Discussion Aedes albopictus is a successful container breeder. Once introduced into an area, its es- tablishment can be considered as evidence. Be- sides, most control efforts focus on the erad- ication of this species in case their first detec- tion attempts prove unsuccessful. Meanwhile, this is dependent on the species’ environ- mental adaptability and the quality of control measures applied (22). Our study was con- ducted in a broad area with imported dengue cases and historical vector collection. Using different mosquitoes sampling methods with- in 12 months, did not obtain any collection of Ae. albopictus. In a similar investigation per- formed in southern California, surveillance of this species after about one year from discov- ery indicated no presence of adults or signs of oviposition. In that research, two types of trap were used: battery-operated CDC/CO2-baited light traps and ovitraps. It was carried out af- ter a report supporting the collection and de- tection of significant numbers of Ae. albopic- tus in the cargo containers of lucky bamboo shipped from China. The rapid control response to the first report of this species and the co- operative efforts aimed at eliminating it were regarded as reasons for this success (22). Mean- while, we do not have any evidence on the eradication efforts carried out by the Iranian health system and their aftereffects on the Ae. albopictus community consequently. Never- theless, some modeling studies have suggest- ed that northern parts of Iran can be suscepti- ble to the establishment of this vector. In a high- resolution map, it was found that enhanced veg- etation index (EVI) annual mean, EVI range, annual monthly maximum precipitation, annu- al monthly minimum precipitation, tempera- ture suitability, and urban as well as peri-ur- ban areas are environmental covariates affect- ing the occurrence probability of Ae. albopic- tus. Specifically, in order of significance, tem- perature suitability, minimum precipitation, and EVI demonstrated the most relative contribu tions to predicting this map (23). Studies con- ducted in southeast of Iran have indicated that low vegetation index, high temperature, along with low rainfall associated with this part of the country affect the establishment of Ae. albopictus in this region (12). In a previous study in this area, Ae. albopictus adults were collected during November/December 2013 after a heavy rainfall (1). Although this spe- cies was not collected in our study, the high- est peak of mosquito activity was preceded by a torrential rainfall. Some researchers attrib- ute an important role to rainfall in establishing Ae. albopictus, such that at least 500 mm of annual rainfall is needed for this species to develop (24, 25). Meanwhile, some studies suggested 290mm of annual rainfall for this to happen (26, 27). Evidently, Ae. albopictus could be established in areas with 200–500 mm annual rainfall (5). Despite the correlation between rainfall and the presence of Ae. Al- bopictus, some researchers have highlighted the behavior of Ae. albopictus as a container mosquito which could exist in the rainfall in- dependently from its breeding sites (28). In a study in India, this species was collected from arid and semi-arid areas. Analogous to the re- gion investigated in the present study, these were plain sandy places with thin vegetation and water stored in containers including a large number of cement and plastic tanks (29). As a result, the risk of Ae. albopictus establishment in this area can never be ignored. Although Ae. albopictus was not collected in the current study, the efficacy of four types of trap on catching six Aedes and Culex species were eval- uated. Some species such as Ae. vexans, Ae. caballus, Cx. quinqaefasciatus, Cx. tritaeniorhyn- chus, and Cx. sitiens can be regarded as West Nile fever and Chikungunya vectors (30-34) that occur in Iran (8, 35). In fact, Cx. quin- qaefasciatus and Ae. vexans have been previ- ously reported as dominant species in south- east of Iran (36, 37). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 385 http://jad.tums.ac.ir Published Online: December 31, 2020 Our results showed that the meteorological factors of wind speed and temperature have a direct significant correlation with the total num- ber of collected mosquitoes. In particular, in southern Norway, mosquito abundance dis- played a negative association with wind speed, and no mosquitoes were collected at speeds above 7·5m/s (38). Our results confirmed that wind speed affects the collection likelihood of several mosquito species.Monitoring densities of mosquito populations can be performed by various sampling methods to evaluate the vec- tors’ behavior, especially the effects of vector control interventions (39). In the current study, CO2-baited bednet trap was associated with the majority of collected mosquitoes. We selected this trap based on a previous study in Iran where it was proved effective in collecting Ae. albopictus (1). Also, this trap has been used for sampling in other vector-borne diseases such as dirofilariasis (40) and malaria (41) in Iran. However, no report has been made so far concerning the efficacy of this trap type for mosquito sampling. Compared to other traps, CO2-baited bednet trap covers a larger space, possibly increasing its ability to collect mos- quitoes especially in a semi-desert climate with low mosquito density. Furthermore, CO2 used in this trap might have served as an efficient mosquito attractant (42). Most studies on trap efficacy have focused on evaluating BG traps versus other traps. For instance, a study in Germany observed that the BG trap was better at collecting a range of mosquito species than three other types of traps: Heavy Duty En- cephalitis Vector Survey trap (EVS trap), Cen- ters for Disease Control miniature light trap (CDC trap) and Mosquito Magnet Patriot Mos- quito trap (MM trap). Also contrary to our find- ings, the widest range of mosquito species was collected by BG traps (43). The field efficacy of the BG trap for collecting of Ae. albopictus has been also studied in New Jersey, US, where it collected more mosquitoes compared with the CDC trap and the gravid trap (GT). There- fore, this trap type has been recommended as an important part of vector monitoring and sur- veillance programs (44). Although most ento- mological studies with BG traps have been un- dertaken in Europe, North and South America, Australia, and Southeast Asia, covering tropi- cal regions or areas with dense vegetation, the poor performance of BG traps in the current study was surprising (44-46). Perhaps this type of trap, especially in the absence of CO2, is less effective in a dry climate with a low mos- quito density. On the other ahnd, Malaise traps (47) and bednet traps performed well in our study. There are few published papers on the usage of Malaise traps for mosquitoes in trop- ical areas of the world (48). In the US, Townes- type Malaise traps (49) were used for about five months to collect mosquitoes, with Aedes species being the most widely collected, fol- lowed by Culex species. Vision as well as dark color can play an important role in the attrac- tion of some insects such as Tabanid flies and mosquitoes (50). Mosquitoes possibly showed a positive response to the Malaise trap due to its black color and its BG lure as an attractant. Since this trap and the bednet traps tend to catch common mosquitoes in similar propor- tions at the same locations and times, both trap types could be useful for general mosquito sur- veys. The so-called resting Bbox trap has been applied and modified for sampling malaria vec- tors, particularly outdoor-resting mosquito spe- cies (39). We modified it to monitor Ae. al- bopictus with a strong exophilic (resting) be- havior (51). However, in our study, this trap did not perform well compared to others. This trap is marked by a low efficiency in collect- ing Cs. inornata (48). There are few published papers regarding the usage of resting box traps for Aedes sampling. In Thailand, two types of trap consisting of short boxes (45cm) and tall boxes (90cm) were placed inside houses (52). The short boxes, whose size was close to the one employed in our trap, attracted relatively more females. Perhaps this type of trap is an efficient method for sampling Ae. aegypti ra- ther than the species collected here. Ovitraps http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 386 http://jad.tums.ac.ir Published Online: December 31, 2020 are commonly used for sampling container- breeding mosquitoes such as Ae. albopictus and Ae. aegypti (53), even though their effi- cacy in catching malaria vectors has also been evaluated (54). In the current study, Ae. al- bopictus larvae were not detected in ovitraps, but the two species of Cx. tritaeniorhynchus and An. stephensi were occasionally observed. In studies designed for mosquito control, Cx. quinquefasciatus, Cx. tritaeniorhynchus, and An. stephensi have been reported alone or along with Ae. aegypti in ovitraps (53, 55-58). Ovit- raps could, therefore, be used in surveillance but their low return rate needs to be taken into account, given that > 500 ovitraps were placed outdoors in this study. Conclusion Data collected here on the efficiency of various trap types can be useful for monitor- ing densities of mosquito populations, which is an important component of a vector surveil- lance system. Although Ae. albopictus had been previously collected from the study area, its establishment is now questionable given that we could not collect it despite substantial ef- fort. Further inquiry into Ae. albopictus estab- lishment in this potential risk area should be rewarding. Eventually, in case of local trans- mission of dengue, studies on the potential transmission of other Aedes species should be considered in any new situation. Acknowledgements This study was financially supported by Tehran University of Medical Sciences with the Grant Number 9121260011 and partially by Ministry of Health and Medical Education under code number of NIMAD 982984. The study was also supported by an NHMRC Fel- lowship to AAH. This research could not be accomplished without the assistance of our col- leagues in Zahedan and Iranshahr health dep- uty. Hence, we would like to express our deep- est appreciation to all those who provided us with the possibility to do this study. We are also grateful to Abdolghafar Hasanzehi and Abdolmohsen Parvin, the health staff in charge of CDC dengue management in Zahedan and Iranshahr Universities of Medical Sciences. We thank Farooq Askani, Aghil Birnoor, Rasulb- akhsh Raeisi, Ebrahim Badroozeh, Salim Nab- atzehi, Shahab Dehghani, and Eng. Noormoham- mad Nabatzehi for their assistance in field sampling. In addition, thanks go to Dr Asghar Talbalaghi, freelance entomologist and consult- ant in IPM in Italy, for his invaluable com- ments during sampling. Last but not least, we thank Eng. Hamid Reza Behjati in Sistan and Baluchestan Meteorological Organization for providing weather data. The authors declare that there is no con- flict of interest. References 1. Doosti S, Yaghoobi-Ershadi MR, Schaffner F, Moosa-Kazemi SH, Akbarzadeh K, Gooya MM, Vatandoost H, Shirzadi MR, Mosta-Favi E (2016) Mosquito sur- veillance and the first record of the in- vasive mosquito species Aedes (Steg- omyia) albopictus (Skuse) (Diptera: Cu- licidae) in Southern Iran. Iran J Public Health. 45(8): 1064–1073 2. Khan J, Munir W, Khan B, Ahmad Z, Shams W, Khan A (2015) Dengue outbreak 2013: Clinical profile of patients pre- senting at DHQ Burner and THQ Shangla, Khyber Pakhtunkhwa, Paki- stan. Immu Dis. 3: 1–4. 3. Mukhtar M, Tahir Z, Baloch TM, Mansoor F, Kamran J (2011) Entomological in- vestigations of dengue vectors in ep- idemic-prone districts of Pakistan dur- ing 2006–2010. Dengue Bull. 35: 99– 115. 4. Suleman M, Faryal R, Aamir UB, Alam MM, Nisar N, Sharif S, Shaukat S, Khurshid A, Angez M, Umair M (2016) Dengue http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 387 http://jad.tums.ac.ir Published Online: December 31, 2020 outbreak in Swat and Mansehra, Pa- kistan 2013: an epidemiological and diagnostic perspective. Asian Pac J Trop Med. 9(4): 380–384. 5. Bueno-Marí R, Jiménez-Peydró R (2015) First observations of homodynamic populations of Aedes albopictus (Skuse) in Southwest Europe. J Vector Borne Dis. 52(2): 175–181. 6. Roiz D, Neteler M, Castellani C, Arnoldi D, Rizzoli A (2011) Climatic factors driving invasion of the tiger mosquito (Aedes albopictus) into new areas of Trentino, northern Italy. PloS One. 6(4): e14800. 7. Talbalaghi A, Moutailler S, Vazeille M, Failloux AB (2010) Are Aedes al- bopictus or other mosquito species from northern Italy competent to sustain new arboviral outbreaks?. Med Vet Ento- mol. 24(1): 83–87. 8. Ardalan M, Chinikar S, Shoja MM (2014) Hemorrhagic Fever with renal syndrome and its history in Iran. Iran J Kidney Dis. 8(6): 438–441 9. Chinikar S, Ghiasi SM, Moradi A, Madihi SR (2010) Laboratory detection facil- ity of dengue fever (df) in iran: the first imported case. Int J Infect Dis. 8(1): 1–2. 10. Aghaie A, Aaskov J, Chinikar S, Niedrig M, Banazadeh S, Mohammadpour HK (2014) Frequency of dengue virus in- fection in blood donors in Sistan and Baluchestan Province in Iran. Transfus Apher Sci. 50(1): 59–62. 11. Nejati J, Saghafipour A, Vatandoost H, Moosa-Kazemi SH, Motevalli Haghi A, Sanei-Dehkordi A (2018) Bionom- ics of Anopheles subpictus (Diptera: Culicidae) in a malaria endemic area, southeastern Iran. J Med Entomol. 55 (5): 1182–1187. 12. Nejati J, Bueno-Marí R, Collantes F, Hanafi- Bojd AA, Vatandoost H, Charrahy Z, Tabatabaei SM, Yaghoobi-Ershadi MR, Hasanzehi A, Shirzadi MR (2017) Po- tential risk areas of Aedes albopictus in south-eastern Iran: a vector of den- gue Fever, Zika and chikungunya. Front Microbiol. 8: 1–12. 13. Urbanelli S, Bellini R, Carrieri M, Sal- licandro P, Celli G (2000) Population structure of Aedes albopictus (Skuse): the mosquito which is colonizing Med- iterranean countries. Heredity. 84(3): 331–337. 14. Pezzin A, Sy V, Puggioli A, Veronesi R, Carrieri M, Maccagnani B, Bellini R (2016) Comparative study on the ef- fectiveness of different mosquito traps in arbovirus surveillance with a focus on WNV detection. Acta Trop. 153: 93– 100. 15. Nejati J, Tabatabaei SM, Salehi M, Sa- ghafipour A, Mozafari E (2017) Some probable factors affecting the malaria situation before and at the beginning of a pre-elimination program in south- eastern Iran. J Parasit Dis. 41(2): 503– 509. 16. Vatandoost H, Emami S, Oshaghi M, Abai M, Raeisi A, Piazzak N, Mahmoodi M, Akbarzadeh K, Sartipi M (2011) Ecol- ogy of malaria vector Anopheles cu- licifacies in a malarious area of Sistan va Baluchestan Province, south-east Is- lamic Republic of Iran. East Mediterr Health J. 17(5): 439–445. 17. Hemami MR, Sari AA, Raeisi A, Vatan- doost H, Majdzadeh R (2013) Malaria elimination in Iran, importance and chal- lenges. Int J Prev Med. 4(1): 88–94. 18. Biogents (2017) Mosquito Trap BG-Sen- tinel 2 Co2 Instruction Manual. Re- gensburg, Germany. 19. Smallegange RC, Schmied WH, van Roey KJ, Verhulst NO, Spitzen J, Mukaba- na WR, Takken W (2010) Sugar-fer- menting yeast as an organic source of carbon dioxide to attract the malaria http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 388 http://jad.tums.ac.ir Published Online: December 31, 2020 mosquito Anopheles gambiae. Malar J. 9(1): 292. 20. Reiter P, Colon M (1991) Enhancement of the CDC ovitrap with hay infusions for daily monitoring of Aedes aegypti populations. J Am Mosq Control Assoc. 7(1): 52–55. 21. Azari-Hamidian S, Harbach RE (2009) Keys to the adult females and fourth- instar larvae of the mosquitoes of Iran (Diptera: Culicidae). Zootaxa. 2078(1): 1–33. 22. Madon MB, Hazelrigg JE, Shaw MW, Kluh S, Mulla MS (2003) Has Aedes albopictus established in California?. J Am Mosq Control Assoc. 19(4): 297– 300. 23. Kraemer MU, Sinka ME, Duda KA, Mylne AQ, Shearer FM, Barker CM, Moore CG, Carvalho RG, Coelho GE, Van Bortel W (2015) The global dis- tribution of the arbovirus vectors Ae- des aegypti and Ae. albopictus. Elife. 4: e08347. 24. Medlock JM, Avenell D, Barrass I, Leach S )2006 ( Analysis of the potential for survival and seasonal activity of Aedes albopictus (Diptera: Culicidae) in the United Kingdom. J Vector Ecol. 31 (2): 292–304. 25. Mitchell C (1995) Geographic spread of Aedes albopictus and potential for in- volvement in arbovirus cycles in the Mediterranean basin. J Vector Ecol. 20(1): 44–58. 26. Benedict MQ, Levine RS, Hawley WA, Lounibos LP (2007) Spread of the tiger: global risk of invasion by the mos- quito Aedes albopictus. Vector Borne Zoonotic Dis. 7(1): 76–85. 27. Severini F, Di ML, Toma L, Romi R (2008) Aedes albopictus in Rome: results and perspectives after 10 years of monitor- ing. Parassitologia. 50(1–2): 121–123. 28. Waldock J, Chandra NL, Lelieveld J, Pro- estos Y, Michael E, Christophides G, Parham PE (2013) The role of envi- ronmental variables on Aedes albopictus biology and chikungunya epidemiology. Pathog Glob Health. 107(5): 224–241. 29. Sarfraz MS, Tripathi NK, Faruque FS, Ba- jwa UI, Kitamoto A, Souris M (2014) Mapping urban and peri-urban breed- ing habitats of Aedes mosquitoes using a fuzzy analytical hierarchical process based on climatic and physical param- eters. Geospat Health. 8(3): S685–S697. 30. Huang YM, Rueda LM (2014) A pictorial key to the species of Aedes (Ochlero- tatus and Coetzeemyia) in the Afrotrop- ical Region (Diptera: Culicidae). Zootaxa. 3754(5): 592–600. 31. Bernard KA, Maffei JG, Jones SA, Kauff- man EB, Ebel G, Dupuis A, Ngo KA, Nicholas DC, Young DM, Shi P-Y (2001) West Nile virus infection in birds and mosquitoes, New York State, 2000. Emerg Infect Dis. 7(4): 679–684. 32. Chapman H, Kay B, Ritchie S, Van den Hurk A, Hughes J (2000) Definition of species in the Culex sitiens subgroup (Diptera: Culicidae) from Papua New Guinea and Australia. J Med Entomol. 37(5): 736–742. 33. Glaser RL, Meola MA (2010) The native Wolbachia endosymbionts of Drosoph- ila melanogaster and Culex quinque- fasciatus increase host resistance to West Nile virus infection. PloS One. 5(8): e11977. 34. Hubalek Z, Halouzka J (1999) West Nile fever, a reemerging mosquito-borne vi- ral disease in Europe. Emerg Infect Dis. 5(5): 643–650. 35. Ahmadnejad F, Otarod V, Fallah M, Low- enski S, Sedighi-Moghaddam R, Zavareh A, Durand B, Lecollinet S, Sabatier P (2011) Spread of West Nile virus in Iran: a cross-sectional serosurvey in equines, 2008–2009. Epidemiol Infect. 139(10): 1587–1593. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 389 http://jad.tums.ac.ir Published Online: December 31, 2020 36. Dehghan H, Sadraei J, Moosa-Kazemi SH (2011) The morphological variations of Culex pipiens (Diptera: Culicidae) in central Iran. Asian Pac J Trop Med. 4(3): 215–219. 37. Yaghoobi-Ershadi M, Doosti S, Schaffner F, Moosa-Kazemi S, Akbarzadeh K, Yaghoobi-Ershadi N (2017) Morpho- logical studies on adult mosquitoes (Diptera: Culicidae) and first report of the potential Zika virus vector Aedes (Stegomyia) unilineatus (Theobald, 1906) in Iran. Bull Soc Pathol Exot. 110(2): 116–121. 38. Hagemoen RIM, Reimers E (2002) Rein- deer summer activity pattern in rela- tion to weather and insect harassment. J Anim Ecol. 71(5): 883–892. 39. Pombi M, Guelbeogo WM, Kreppel K, Calzetta M, Traoré A, Sanou A, Ran- son H, Ferguson HM, Sagnon NF, Della Torre A (2014) The Sticky Rest- ing Box, a new tool for studying rest- ing behaviour of Afrotropical malaria vectors. Parasit Vectors. 7(1): 247. 40. Azari Hamidian S, MR YE, Javadian E, Abai M, Mobedi I, Linton YM, Har- bach R (2009) Distribution and ecol- ogy of mosquitoes in a focus of diro- filariasis in northwestern Iran, with the first finding of filarial larvae in natu- rally infected local mosquitoes. Med Vet Entomol. 23(2): 111–121. 41. Moosa-Kazemi SH, Firoozfar F (2016) Bi- onomic studies of the mosquitoes (Dip- tera: Culicidae) in Kermanshah Prov- ince, Western Iran. Life Sci J. 13(10): 50–55. 42. Gillies M (1980) The role of carbon diox- ide in host-finding by mosquitoes (Dip- tera: Culicidae): a review. Bull Ento- mol Res. 70(4): 525–532. 43. Lühken R, Pfitzner WP, Börstler J, Garms R, Huber K, Schork N, Steinke S, Kiel E, Becker N, Tannich E (2014) Field evaluation of four widely used mos- quito traps in central Europe. Parasit Vectors. 7(1): 268. 44. Farajollahi A, Kesavaraju B, Price DC, Williams GM, Healy SP, Gaugler R, Nelder MP (2009) Field efficacy of BG-sentinel and industry-standard traps for Aedes albopictus (Diptera: Cu- licidae) and West Nile virus surveil- lance. J Med Entomol. 46(4): 919–925. 45. Ritchie SA, Moore P, Carruthers M, Wil- liams C, Montgomery B, Foley P, Ahboo S, Van Den Hurk AF, Lindsay MD, Cooper B (2006) Discovery of a widespread infestation of Aedes al- bopictus in the Torres Strait, Australia. J Am Mosq Control Assoc. 22(3): 358–365. 46. Hoffmann A, Montgomery B, Popovici J, Iturbe-Ormaetxe I, Johnson P, Muzzi F, Greenfield M, Durkan M, Leong Y, Dong Y (2011) Successful establish- ment of Wolbachia in Aedes popula- tions to suppress dengue transmission. Nature. 476(7361): 454–461. 47. Malaise R (1937) A new insect-trap. Ento- mol Tidskr. 58: 148–160. 48. Service M (1993) Mosquito Ecology Field Sampling Methods. Springer, Switzer- land. 49. Townes H (1962) Design for a Malaise trap. Proc Ent Soc Wash. 64: 253–262. 50. Van Breugel F, Riffell J, Fairhall A, Dick- inson MH (2015) Mosquitoes use vi- sion to associate odor plumes with ther- mal targets. Curr Biol. 25(16): 2123– 2129. 51. Delatte H, Desvars A, Bouétard A, Bord S, Gimonneau G, Vourc'h G, Fon- tenille D (2010) Blood-feeding behav- ior of Aedes albopictus, a vector of Chikungunya on La Réunion. Vector Borne Zoonotic Dis. 10(3): 249–258. 52. Edman J, Kittayapong P, Linthicum K, Scott T (1997) Attractant resting boxes for rapid collection and surveillance of Aedes aegypti (L.) inside houses. J Am http://jad.tums.ac.ir/ J Arthropod-Borne Dis, December 2020, 14(4): 376–390 J Nejati et al.: Employing Different Traps for … 390 http://jad.tums.ac.ir Published Online: December 31, 2020 Mosq Control Assoc. 13(1): 24–27. 53. Ritchie SA, Long S, Hart A, Webb CE, Russell RC (2003) An adulticidal sticky ovitrap for sampling container-breed- ing mosquitoes. J Am Mosq Control Assoc. 19(3): 235–242. 54. Elango G, Zahir AA, Bagavan A, Kamaraj C, Rajakumar G, Santhoshkumar T, Marimuthu S, Rahuman AA (2011) Ef- ficacy of indigenous plant extracts on the malaria vector Anopheles subpic- tus Grassi (Diptera: Culicidae). Indian J Med Res. 134(3): 375–379. 55. Surendran SN, Jude PJ, Thavaranjit AC, Eswaramohan T, Vinobaba M, Rama- samy R (2013) Predatory efficacy of Culex (Lutzia) fuscanus on mosquito vectors of human diseases in Sri Lanka. J Am Mosq Control Assoc. 29(2): 168– 170. 56. Elizondo-Quiroga A, Flores-Suarez A, Elizondo-Quiroga D, Ponce-Garcia G, Blitvich BJ, Contreras-Cordero JF, Gon- zalez-Rojas JI, Mercado-Hernandez R, Beaty BJ, Fernandez-Salas I (2006) Gonotrophic cycle and survivorship of Culex quinquefasciatus (Diptera: Cu- licidae) using sticky ovitraps in Mon- terrey, northeastern Mexico. J Am Mosq Control Assoc. 22(1): 10–14. 57. Seenivasagan T, Sharma KR, Prakash S (2012) Electroantennogram, flight ori- entation and oviposition responses of Anopheles stephensi and Aedes aegypti to a fatty acid ester-propyl octade- canoate. Acta Trop. 124(1): 54–61. 58. Hesson JC, Ignell R, Hill SR, Östman Ö, Lundström JO (2015) Trapping biases of Culex torrentium and Culex pipiens revealed by comparison of captures in CDC traps, ovitraps, and gravid traps. J Vector Ecol. 40(1): 158–163. http://jad.tums.ac.ir/