Yu-Chen Chen, Chih-Yuan Wang, Hwa-Jen Teng, Chien-Fu Chen, Mi-Chun Chang, Liang-Chen Lu, Cheo Lin, Shu-Wan Jian, and Ho-Sheng Wu

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1 68 Journal of Vector Ecology June 2011 Comparison of the efficacy of -baited and unbaited light, gravid, backpack aspirators, and sweep net collections for sampling mosquitoes infected with Japanese encephalitis virus Yu-Chen Chen, Chih-Yuan Wang, Hwa-Jen Teng, Chien-Fu Chen, Mi-Chun Chang, Liang-Chen Lu, Cheo Lin, Shu-Wan Jian, and Ho-Sheng Wu Research and Diagnostic Center, Centers for Disease Control, Taiwan, ROC Received 13 July 2010; Accepted 2 December 2010 ABSTRACT: Two field studies were conducted to determine the efficacy of mosquito collection methods for species composition, species abundance, and Japanese encephalitis virus infection rates in Taiwan. Traps evaluated included John W. Hock (JH) model UD black light, JH model 1012 new standard miniature CDC light, JH model 1712 CDC gravid, and Taiwan-made Pest-O-Lite light. Backpack aspirators and sweep nets were also used to collect the resting population. Culex tritaeniorhynchus in all studies and Mansonia uniformis in the Taipei areas were the two most abundance species collected. Dry ice-baited UD black light were effective in regard to species diversity, species abundance, and Japanese encephalitis virus infection rates. The unbaited Pest-O-Lite light collected significantly more female mosquitoes than the UD black light but performed similarly with regard to species diversity and male mosquito collection. Most mosquitoes collected by Pest-O-Lite light were dried and not suitable for virus detection. Dry icebaited CDC light collected significantly fewer mosquitoes than other light. Although -baited UD black light with octenol attracted more mosquitoes, no statistical significance was found compared to -baited UD black light without octenol. Japanese encephalitis viruses were isolated from half of the positive in UD black light and CDC light. Journal of Vector Ecology 36 (1): Keyword Index: Light, Culex tritaeniorhynchus, JE virus. INTRODUCTION Of the 132 mosquito species that have been identified in Taiwan, Aedes aegypti (L.), Ae. albopictus (Skuse), Culex tritaeniorhynchus Giles, Cx. annulus Theobald, Cx. fuscocephalus Theobald, and Anopheles minimus Theobald are major local vectors of dengue, Japanese encephalitis, and malaria. Among these diseases, only Japanese encephalitis is considered to be an endemic disease in Taiwan, while the other two diseases are at present considered to be travelrelated diseases (Wu et al. 2009). Mosquito surveillance to determine species composition, species abundance, and pathogen infection rates provides basic information for control strategies and measures of mosquito-borne infectious diseases. Surveillance requires accurate sampling techniques of adult mosquitoes based on the behavior and ecology of the target species. Common techniques used to collect adult mosquitoes include human landing collections, light, gravid, backpack aspirators, and sweep nets. However, each of these techniques has drawbacks that interfere with its effectiveness and accuracy. Human landing collection, backpack aspirators, and sweeping nets are unfavorable due to their labor-intensiveness, collector factors, and the risk of exposing collectors to pathogen transmission. Light attract most mosquitoes but might miss some nonphototactic Culex species (Tsai et al. 1988, Nayar et al. 2001, Kline et al. 2006). A widely used light trap for the surveillance of vector mosquitoes of West Nile virus is the Centers for Disease Control (CDC) trap, which uses carbon dioxide and a light source to attract mosquitoes. Furthermore, most mosquitoes collected by this trap type are host-seeking mosquitoes that include large quantities of first-time feeders that will underestimate the infection rates of pathogens. An alternative to the light trap for Culex collection is the gravid trap, which is designed to collect gravid female mosquitoes that are attracted to a potential oviposition site containing water high in organic matter (Reiter 1983, 1987). Hence, it assures that the collected mosquitoes blood-fed at least once, increasing the possibility of detecting viral infections in the mosquito population. The addition of olfactory attractants in light has been found to be effective in the collection of host-seeking mosquitoes in some but not all species (Canyon and Hii 1997, Mboera et al. 2000, McCardle et al. 2004, Shone et al. 2006, Muturi et al. 2007). These attractants include, lactic acids, 1-octen-3-ol, acetone, and carboxylic fatty acids (Takken and Knols 1999), and serve as kairomones or allomones. The most commonly used chemicals were and octenol, which are considered as long range kairomones. A kairomone is a semiochemical or infochemical that acts between individuals of different species (hosts) to the benefit of the receiver (mosquitoes). Carbon dioxide appears to be universally attractive to mosquitoes (Gibson and Torr 1999). 1-octen-3-ol is a chemical compound present in bovine emanations, which especially attracts various Culex,

2 Vol. 36, no. 1 Journal of Vector Ecology 69 Anopheles, and Aedes species, especially when combined with (Kline 1994). Many of these species are also important vectors of mosquito-borne diseases. Therefore, trapping systems that incorporate combinations of these attractants may enhance surveillance for these diseases and provide a better estimate of disease prevalence in individual vector species. Mosquito surveillance for estimating abundance and infection rates is problematic. In Taiwan, we used unbaited Pest-O-Lite light hung in front of a house to estimate the distribution and abundance of An. minimus. The collected mosquito samples were usually dried, damaged, and not suitable for virus detection. Therefore, finding a trap for collecting suitable mosquito samples to determine both species abundance and infection rates is highly desirable for mosquito surveillance. The objective of our study was to compare different collection methods with and without attractants for species diversity, species abundance, and virus infection rates in mosquitoes to determine which trap is the most effective for mosquito surveillance in Taiwan. MATERIALS AND METHODS Study sites Trapping studies were conducted at human dwellings of rural areas in southeast Taiwan and in natural wild fields (a natural park and an animal zoo) in Taipei in northern Taiwan. At the wild field sites, there were ponds and birds. The villages of southeastern Taiwan have rice fields and livestock in close quarters to human dwellings. Evaluation of light in villages Two light, the UD black light trap (Model 1312, John W. Hock Company, Gainesville, FL) and the Pest-O-Lite light trap (locally manufactured in Taiwan), were evaluated in this study. The UD black light trap uses a 4-W black light tube ( nm) and a 6 VDC battery, whereas the Pest- O-Lite light trap (18.5 cm dia. base and 28 cm dia. cover x 25 cm high) also uses a black light ( nm) but with an AC electric plug. Mosquitoes are attracted by the black light on the top cover and captured by a vacuum mechanism downward into a net bag. From April to September, 2005 and 2006, two to three villages were surveyed each month in southeast Taiwan for a total of 19 villages. At each visit, these two were set up outside of the houses overnight approximately m off the ground. On the same day, two teams collected adult mosquitoes inside the houses and their surroundings for one hour between 15:00 and 17:00. Each team included two people with one modified CDC backpack aspirator (Model 1412, John W. Hock Company) and one sweep net. Additionally, the effect of octenol paired with -baited UD black light was evaluated from June to September, 2006 inside the villages, village edge, and outside the villages. Evaluation of light and gravid in open fields Three types of, UD black light trap (Model 1312, the same as above), new standard miniature light trap (Model 1012, John Hock Company), and gravid trap (Model 1712, John Hock Company) were evaluated in this study. The new standard miniature CDC light trap uses an incandescent light (CM47 bulb) source and requires a 6 V battery. Both light were baited with dry ice and were hung from the trees approximately 1.5 m off the ground. The gravid, collecting gravid females, were placed on the ground in partially shaded areas. Mosquitoes are drawn into the gravid from the water surface into a bag by a 6 V fan positioned inside a PVC intake tube which is suspended over a grey pan (22 cm in width x 34 cm in length x 17 cm in depth) containing an oviposition attractant. A yeast infusion (1 kg to 1 liter H 2 O) incubated for seven days was chosen based as the oviposition attractant based on a previous pilot study. Each month, these three were set up randomly overnight from 17:00 to 09:00 the next morning at two sites in the Nature Park and the Animal Zoo, respectively, from April-August, Additionally, human collections by sweeping nets were conducted to collect the mosquitoes on the same day between 17:30 and 19:30 for 30 min. Virus detection in mosquitoes All mosquitoes collected from 2005 to 2008 by various methods were stored on dry ice and brought back to the laboratory for species identification and stored at 20 o C for virus detection. A one-step SYBR Green I-based Real-Time Reverse Transcription-PCR Assay was used to test for mosquito Japanese Encephalitis infection rates. The detailed protocol of this method was performed as described by Yang et al. (2010). Mosquito were homogenized and clarified by centrifugation. Viral RNAs (70 μl) were extracted from 140 ml of mosquito suspension using the QIAamp viral RNA mini kit (cat. no. 52,906, Qiagen, Hilden, Germany) according to the manufacturer s instructions. RT-PCR amplification was performed in the Mx4000 quantitative PCR system (Stratagene, CA, U.S.A.). Samples were assayed in a 50 μl reaction mixture containing 10 μl of sample RNA and optimal concentration of the primers by using QuantiTect SYBR Green RT-PCR kit (cat. no. 204, 243, Qiagen, Hilden Germany). The thermal profile consisted of a 30-min RT step at 50 C and 15 min of Taq polymerase activation at 95 C, followed by 45 cycles of PCR. Following amplification, a melting curve analysis was performed to verify the correct product by its specific melting temperature. Furthermore, positive PCR products were sequenced in order to determine disease profile. The maximal number of mosquitoes tested per pool was 50. All positive of mosquitoes were subjected to virus isolation. Statistical analysis All statistical analyses were performed using Statistica version 5.0 statistical package (StatSoft Holdings, Inc., Tulsa, OK, U.S.A.). Because the main objective of the study was to compare the effectiveness of the and not the difference between sites, data for each site was treated as a block to eliminate site variance. The species diversity and mosquito abundance were analyzed by a completely

3 70 Journal of Vector Ecology June 2011 randomized block design. Statistical analysis was done after log transformation of adult mosquito counts to normalize the distribution and minimize the standard error. Means separation was made using a least significant difference test. Additionally, a paired t-test was used to compare the differences in the mean number of mosquitoes collected by -baited UD black light with and without octenol. RESULTS Evaluation of light in villages Significantly more species (P < 0.01), more females (P < 0.01), and fewer males (P < 0.05) were collected in light than by using backpack aspirators (Table 1). For species diversity, UD black light (5.4 species per trap) and Pest-O-Lite light (4.8 species per trap) were significantly better than backpack aspirators (2.9 species per trap). For female abundance, Pest-O-Lite light (77.3 females per trap) were best followed by UD black light (49.6 females per trap) and backpack aspirators (15.8 females per trap). For male abundance, backpack aspirators (16.8 males per trap) collected significantly more males than the Pest-O-Lite and UD black light (7.7 and 5.1 males per trap). From a total of 1,614 mosquitoes that were collected during the sampling period, 19 mosquito species from the genera Aedes, Anopheles, Armigeres, Conquillettidia, Culex, and Mansonia were captured in Pest-O-Lite light (Table 2). Culex tritaeniorhynchus constituted 45.2% of the total collection followed by Ar. subalbatus (22.2%), An. sinensis (13.1%), An. ludlowae (5.8%), An. minimus (3.1%), Cx. quinquefasciatus (2.8%), Cx. annulus (2.0%), An. maculatus (1.3%), and Cx. sitens (1.2%). The other ten species occurred in quantities smaller than 1% of the total collection. In the -baited UD black light, 23 species were captured. The most abundant species in these collections was Cx. tritaeniorhynchus (24.4%), followed by Ar. subalbatus (18.3%), Ae. vexans vexans (14.5%), Ae. albopictus (10.6%), An. ludlowae (6.1%), Cx. quinquefasciatus (5.0%), Cx. annulus (4.5%), An. minimus (4.2%), An. maculatus (2.9%), Ae. desmotes (2.5%), An. sinensis (2.3%), and Cx. sitiens (1.9%). The other 11 species were represented in quantities lower than 1% of the total capture. In backpack aspirators, only seven species were captured. The most abundant species was Ar. subalbatus (49.4%), followed by Cx. quinquefasciatus (30.9%), and Ae. albopictus (18.1%). The other four species were captured in quantities lower than 1%. In addition, the effect of octenol was also evaluated at the same sites. Although octenol (in addition to in UD black light ) attracted more mosquitoes (males, females, and total), no statistically significant difference was found (P > 0.05). The mean number of species, males, females, and total attracted were 3.4 species, 4.8 males, 57.5 females and 62.3 mosquitoes per trap in -baited UD black light, and 4.6 species, 7.1 males, 87.4 females, and 94.5 mosquitoes in -baited UD black light paired with octenol, respectively. Evaluation of light and gravid in open fields Significant differences among the five mosquitocollecting methods were found in respect to species diversity, mosquito abundance, female quantity, and male quantity (P < 0.01) (Table 3). For species diversity, light (7.7, 5.6, and 5.9 species per trap in downward or upward -baited UD black light and CDC light, respectively) collected significantly more species than gravid (1.6 species per trap) and sweeping nets (3.1 species per trap). For mosquito abundance, significantly more mosquitoes were collected by light ( mosquitoes per trap-night) than sweeping nets (47.4 mosquitoes per night) and gravid (11.3 mosquitoes per trap-night). Higher numbers of female mosquitoes were collected in light ( females per trap-night) than sweeping net (31.9 females per night) and gravid (9.7 females per trap-night). Significantly more males were collected by sweeping nets (13.5 per trap-night) than light ( per trap-night). From a total of 13,124 mosquitos that were collected during the sampling period, twenty-seven mosquito species from the genera Aedes, Anopheles, Armigeres, Conquillettidia, Culex, Heizmannia, Mansonia, Tripteroides, and Uranotaenia were captured (Table 4). In UD black light, Culex tritaeniorhynchus constituted % of the total collection in UD black light, Table 1. Numbers of mosquito species, males, and females collected in backpack aspirators, Pest-O-Lite light, and UD black light in human dwellings from 2004 to Variables n Pest-O-Lite light trap Collection methods UD black light Backpack aspirator trap Species a 5.4 a 2.9 b 5.8 <0.01 Males a 5.1 a 16.8 b 9.2 <0.01 Females a 49.6 b 15.8 c 4.3 < a 54.7 a 32.6 a 3.0 >0.05 Means within each row (species, sex, and total) having the same letter are not significantly different at P F 2,36 P

4 Vol. 36, no. 1 Journal of Vector Ecology 71 Table 2. number (percentage) of mosquito species caught in various collecting methods in villages of southern Taiwan from 2004 to Mosquito species Pest-O-Lite light -baited UD black light Backpack aspirator Cx. tritaeniorhynchus 730 (45.2) 254 (24.4) 6 (1.0) Ar. subalbatus 358 (22.2) 190 (18.3) 306 (49.4) An. sinensis 213 (13.2) 24 (2.3) 0 An. ludlowae 94 (5.8) 63 (6.1) 0 An. minimus 50 (3.1) 44 (4.2) 1 (0.2) Cx. quinquefasciatus 45 (2.8) 52 (5.0) 191 (30.9) Cx. annulus 33 (2.0) 47 (4.5) 2 (0.3) An. maculatus 21 (1.3) 30 (2.9) 1 (0.2) Cx. sitiens 20 (1.2) 20 (1.9) 0 Ae. albopictus 13 (0.8) 110 (10.6) 112 (18.1) Cx. fuscocephala 11 (0.7) 4 (0.4) 0 Ae. vexans vexans 6 (0.4) 151 (14.5) 0 Other species* 20 (1.2) 51 (4.9) 0 1,614 (100.0) 1,040 (100.0) 619 (100.0) *14 species included Aedes desmotes, Ae. lineatopennis, Ae. albolateralis, Anopheles tessellates, Anopheles species, Armigeres omissus, Coquillettidia crassipes, Culex bitaeniorhynchus, Cx. fuscanus, Cx. hayashii, Cx. neomimulus, Cx. nigropunctatus, Cx. culiciomyia sp., and Mansonia uniformis. Table 3. Mean number of mosquitoes collected in UD black light, CDC light, and gravid during April- August Variable n -baited UD black light Downward Upward - baited CDC light Gravid Sweeping nets Species a 5.6a 5.9a 1.6b 3.1b 7.6 <0.01 Males a 4.3a 5.0a 1.5a 13.5b 3.8 <0.01 Females a 129.5a 114.4ab 9.7c 31.9bc 4.8 < a 133.8a 119.3a 11.3b 47.3b 4.6 <0.01 Means within each row (species, sex, and total) having the same letter are not significantly different at P F 4, 88 P

5 72 Journal of Vector Ecology June 2011 Table 4. number (percentage) of mosquito species caught in various collecting methods in Taipei, Taiwan, during April-August, Mosquito species Downward -baited UD black light Upward -baited UD black light -baited CDC light Gravid Sweep net Culex tritaeniorhynchus 2,941 (66.7) 2,264 (73.6) 982 (35.8) 13 (4.4) 263 (24.2) Mansonia uniformis 964 (21.9) 512 (16.6) 1,378 (50.2) 189 (64.1) 425 (39.0) Cx. annulus 148 (3.4) 140 (4.5) 158 (5.8) 1 (0.3) 7 (0.6) Aedes albopictus 109 (2.5) 47 (1.5) 132 (4.8) 22 (7.5) 245 (22.5) Cx. mimulus 60 (1.4) 7 (0.2) 14 (0.5) 0 (0.0) 6 ( 0.6) Cx. rubithoracis 56 (1.3) 42 (1.4) 17 (0.6) 3 (1.0) 13 (1.2) Cx. quinquefasciatus 47 (1.1) 22 (0.7) 20 (0.7) 13 (4.4) 89 (8.2) Other species* 84 (1.9) 36 (1.2) 44 (1.6) 18 (6.1) 41 (3.8) 4,409 (100.0) 3,078 (100.0) 2,745 (100.0) 295 (100.0) 1,089 (100.0) *19 species included Aedes penghuensis, Ae. vexans vexans, Anopheles sinensis, An. tessellates, Armigeres subalbatus, Coquillettidia crassipes, Culex bicornutus, Cx. bitaeniorhynchus, Cx. fuscanus, Cx. halifaxii, Cx. infantulus, Cx. murrelli, Cx. pallidothorax, Cx. pseudovishnui, Heizmannia taiwanensis, Mimomyia luzonensis, Tripteroides bambusa, Tr. aranoides, and Uranotaenia niviplura. Table 5. Japanese Encephalitis virus infection in field-collected mosquitoes in Taiwan by various collection methods, Mosquito species UD black light CDC light PO Lite light ** Gravid * Human collection and sweeping nets ( rate %) Cx. tritaeniorhynchus (6) 61 2 (1) 89 1 (0) (6) 750 (3.3) Cx. fuscocephalus (0) (4.3) Cx. annulus (0.0) Other species ,145 (0.0) 1, (6) (1) (0) (6) 2,040 (1.3) *Up to 50 mosquitoes per pool. ** Mosquitoes tested were selected under a microscope.

6 Vol. 36, no. 1 Journal of Vector Ecology 73 followed by Ma. uniformis ( %), Cx. annulus ( %), and Ae. albopictus ( %). The remaining 21 species occurred in small quantities. In CDC light, 22 species were captured. The most abundant species in these collections were Ma. uniformis (50.2%), followed by Cx. tritaeniorhynchus (35.8%), Cx. annulus (5.8%), and Ae. albopictus (4.8%). The remaining 17 species were represented in quantities lower than 1% of the total capture. In gravid, only 11 species were captured. The most abundance species were Ma. uniformis (64.1%), followed by Ae. albopictus (7.5%), Cx. tritaeniorhynchus (4.4%), and Cx. quinquefasciatus (4.4%). The remaining seven species were represented in small quantities. In sweeping nets, 17 species were collected. The most prevalent species were Ma. uniformis (39.0%), Cx. tritaeniorhynchus (24.2%), and Ae. albopictus (22.5%), followed by Cx. quinquefasciatus (8.2%) and Ar. subalbatus (1.6%). Virus detection A total of 39,295 mosquito adults (17,968 females and 644 males) were grouped by species, place, and date into 2,040 (Table 5). Overall, only 3.3% of the 750 of Cx. tritaeniorhynchus (28,202 females and 63 males) and 4.3% of the 23 of Cx. fuscocephalus (349 females and 63 males) were found to be positive for Japanese Encephalitis virus by RT-PCR. Twelve positives were collected from UD black light and two positives were collected from CDC light. The highest virus isolation rate for positive was 60.0% for mosquitoes collected by human collection with collection tubes and sweeping nets, followed by mosquitoes collected in UD black light and CDC light (50.0%). No isolation was successful for mosquitoes collected in Pest-O-Lite light. DISCUSSION The efficacy of different mosquito collection methods varied in terms of species diversity, species abundance, and Japanese Encephalitis virus infection rates. According to our trap data, the -baited UD black light were efficient for surveillance of species diversity, species abundance, and Japanese Encephalitis virus infection rates. The unbaited Pest-O-Lite light trap was also efficient for surveillance of mosquito species diversity and species abundance but not for virus infection rates. -baited CDC light also worked well in regard to species diversity (Ma. uniformis, Cx. tritaeniorhynchus, Cx. annulus, and Ae. albopcitus). The yeast-baited gravid trap were fair in collecting mostly Ma. uniformis. In our studies, Cx. tritaeniorhynchus and Ma. uniformis were the most abundant species. The former species is a cosmopolitan mosquito occurring in a wide range of larval habitats, including vegetated lakes and rivers, natural ponds, rice fields, agricultural ponds, and temporary. This species, an opportunist, may feed on pigs, humans, and birds and is highly attracted to lights. In our study, few Cx. quinquefasciatus were collected in light and gravid compared to backpack aspirators and sweeping nets. In general, mosquitoes respond to the using visual, olfactory, and tactile cues. The light used in this study attracted females with visual cues with and without olfactory cues from, whereas gravid attract with olfactory cues from ovipositional attractants. However, other visual cues, such as pan color, pan size, and water color of the gravid trap are also important in the orientation of mosquitoes in oviposition behavior in a variety of mosquito species (Allan and Kline 2004, Allen et al. 1987, Beehler et al. 1993, Bentley and Day 1989). Oviposition by Cx. quinquefasciatus occurs under low light conditions, typically with most activity within two hours after sunset (Nayar 1982, Schreiber et al. 1989, Beehler et al. 1993). In our study, most positive were found in Cx. tritaeniorhynchus females, which were captured on June through July by UD black and CDC light. 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