1. Introduction

Lymphatic filariasis is a vector-borne nematode infection caused by Wucherea bancrofti, Brugia malayi and Brugia timori, and more than 100 million people are affected worldwide [1,2]. Of this total, 90% are caused by W bancrofti and 10% by B malayi [3]. The first case of elephantiasis in Korea was reported in 1927 [4], and it was called by various names: Soojongdari (leg dropy), Pinaerim (blood down flow), Pijoeng (blood diseases), and Gakmomsal (malaise with arthralgia) [5]. In the 1940s, the infection rate reached as high as 12–26% in the southern parts of the country and Jeju-do [6-8]. The rate remained at a 12% average until the early 1970s (range, between 5.5% and 18.0%) in the more inland areas of Youngju-si (si = district), Gyeongsangbuk-do (do = province). Following the government’s active efforts for treatment, the disease was eliminated in the late 1980s [9-11]. By the mid-1980s, the average rate fell below 1% even in Jeju-d,o where the infection was most prevalent [12]. Furthermore, no positive cases of filariasis were reported in past endemic areas of Jeollanam-do, Gyeongsan-do, and Jeju-do between 2002 and 2006 [13]. Finally, the World Health Organization verified that filariasis was eliminated in Korea, declaring the country free from lymphatic filariasis [13-15]. However, monitoring of vector mosquitoes should continue to prevent possible reemergence due to increasing overseas travel and climate changes. In this study, we investigated the density and distribution of

Figure 1.

Area surveyed for adult and immature mosquitoes in 2009.

vector mosquitoes of filariasis, and its DNA from vector mosquitoes was tested.

2. Materials and Methods

2.1. Survey areas

Jeollanam-do [Sa-ri (ri = village), Heuksan Island in Shinan-gun, Geomun-ri, Geomun Island in Yeosu-si (si = district), Baekdo-ri, Bogil Island in Wando-gun], Gyeongsangnam-do (Maejuk-ri, Maemul Island in Tongyoung-si), and Jeju-do (Island) (Wimi-ri, Seoguipo-si) were surveyed (Figure 1).

2.2. Adult mosquito surveillance

Adult mosquitoes were collected twice weekly using the commercial Black Hole® mosquito black light trap (Model: Black Hole, Bio-trap Inc., Seoul, Korea). The traps were placed under the eaves of houses from 19:00 to 06:00 hours the following morning. All mosquitoes were identified to the genus or species level under a dissecting microscope using standard morphological keys [16,17]. An sinensis was assigned to the Anopheles (Hyrcanus) group, because of the difficulty in identifying the species by microscope.

2.3. DNA extraction and PCR condition

The selected Och togoi and An (Hyrcanus) Group were pooled to 1–3 mosquitoes, and total DNA was extracted with the QIAamp DNA kit according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). Hha1 F 5'-

Table 1.

Seasonal variations of mosquitoes collected in mosquito trap in Sa-ri, Heuksan lsland (latitude 34°39'N, longitude 125°25'E), Shinan-gun, Jeollanam-do in 2009

Month	An (Hyicanus) group	Arm sabalbatus	Cx Bitaenior	Cx pipiens	Cx tritaenior	Och togoi	Mean no./trap
May		1				7	8
Jun		1		1		19	21
Jul	1	3				14	18
Aug		2		3	6	22	33
Sep		6	1	8	2	19	36
Oct		7		13	1	12	33
Total (%)	1 (0.7)	20 (13.4)	1 (0.7)	25 (16.8)	9 (6)	93 (62.4)	149

Numbers in parenthesis represent data on male mosquitoes.

GCGCATAAATTCATCAGC-3', R 5'-GCGCAAAAC TTAATTACAAAAGC-3' of two set primers were used [18]. Polymerase chain reaction (PCR) was performed in a 25-μL reactive solution containing 5 μL of DNA template, 20 pmol of each primer, 1.25 U of ExTaq DNA polymerase (Takara Co., Japan), 2 mM MgCl₂, and 250 μM of each dNTP. The following PCR cycle was performed in an iCycler thermal cycler (Bio-Rad Ltd., Hercules, CA, USA): 1 × 94℃ for 5minutes, 40 × (94℃ for 1 minute, 56℃ for 1 minute, 72℃ for 1 minute), 1 × 72℃ for 10 minutes.

3. Results

3.1. Sa-ri, Heuksan lsland, Shinan-gun

A total of 149 mosquitoes were collected in Heuksan Island, consisting of four genera and six species. Among them, Och togoi was the most common (62.4%), followed by Culex pipiens (16.8%), Armigeres subalbatus (13.4%), Culex tritaeniorhynchus (6%), Culex bitaenirohynchus (0.7%), and An (Hyrcanus) Group (0.7%) (Table 1). However, filariasis DNA was not detected in the vector species of Och togoi and An (Hyicanus) group.

3.2. Baekdo-ri, Bogil lsland, Wando-gun

In Bogil Island, 3097 mosquitoes were collected in seven genera and 12 species. Och togoi was the most

Table 2.

Seasonal variations of mosquitoes collected in mosquito trap in Baekdo-ri, Bogil lsland (latitude 34°08'N, longitude 126°32'E), Wando-gun, Jeollanam-do, in 2009

Month	Ae vexans	An (Hyicanus) group	Arm sabalbatus	Cog ochracea	Cx Bitaenior	Cx pipiens	Cx tritaenior	Och dorselis	Och nipponicus	Och togoi	Lutrie vorax	Mean no./trap
May		4	1			3		4		19		31
Jun		30	5			3		10		318		366
Jul		23	16		1	6	39	12	1	737		835
Aug		106	21	9		16	77	9	2	1,005		1,245
Sep	1	31	35			13	12		1	527	1	620
Total	1 (0.03)	164 (6.3)	78 (2.5)	9 (0.3)	1 (0.03)	41 (1.3)	128 (4.1)	35 (1.1)	4 (0.1)	2,606 (84.1)	1 (0.03)	3,097
(%)

Numbers in parenthesis represent data on male mosquitoes.

frequent (84.1%), followed by An (Hyrcanus) Group (5.3%), Cx tritaeniorhynchus (4.1%), Ar subalbatus (2.5%), Cx pipiens (1.3%), Och dorsalis (1.1%), An sinesis (1.0%), Coh ochracea (0.3%), Och nipponicus (0.1%), Ae vexans (0.03%), and Lutrie vorax (0.03%). The number of collected Och togoi rose sharply between May and June, and remained high until September. Neither Och Togoi nor An (Hyicanus) group had filariasis DNA (Table 2).

3.3. Geomun-ri, Geomun lsland, Yeosu-si

A total of 376 mosquitoes were collected in Geomunri, Geomun Island, comprising five genera and eight species. Among them, the number of Cx pipiens was highest (45.2%), followed by Och togoi (42%), Ar subalbatus (6.4%), Cx triraenior (4.8%), Ae albopirtus (0.8%), Cx inatomi (0.3%), Och koreicus (0.3%), and Lu vorax (0.3%) (Table 3). Filariasis DNA was not found in Och togoi and An (Hyicanus) group.

3.4. Maejuk-ri, Maemul lsland, Tongyoung-si

A total of 1213 mosquitoes were collected in Maejuk-ri, Maemul Island, comprising of four genera and seven species. Och togoi was most frequently caught (84.9%), followed by Ar subalbatus (12%), Cx pipiens (2.1%), Cx triraenior (0.5%), Ae albopirtus (0.3%), Och koreicus (0.2%), and Cx bitaenior (0.1%). The number of Och togoi picked up sharply between May and June, and dropped significantly between

Table 3.

Seasonal variations of mosquitoes collected in mosquito trap in Geomun-ri (latitude 34°17'N, longitude 127°23'E), Geomun lsland, Yeosu-si, Jeollanam-do in 2009

Month	Ae albopirtus	Arm sabalbatus	Cx inatomi	Cx pipiens	Cx tritaenior	Och koreicus	Och. togoi	Lutrie. vorax	Mean no./trap
May				1			10		11
Jun		2	1	20	12	1	31		67
Jul	2	6		40	4		52		104
Aug	1	6		17			18		43
Sep		3		33			28	1	65
Oct		7		59	2		18		86
Total (%)	3 (0.8)	24 (6.4)	1 (0.3)	170 (45.2)	18 (4.8)	1 (0.3)	158 (42)	1 (0.3)	376

Numbers in parenthesis represent data on male mosquitoes.

September and October (Table 4). Filariasis DNA was not found in Och togoi and An (Hyicanus) group.

3.5. Namone-ri, Seoguipo-si

A total of 247 mosquitoes were collected in Namoneri, Seoguipo-si, Jeju-do, comprising two genera and four species. Cx pipiens was most frequently caught (81.8%), followed by Och togoi (13%), Cx triraenior (4.9%), and Cx mineficus (0.4%). No filariasis DNA was detected in Och togoi and An (Hyicanus) group (Table 5).

3.6. Wimi-ri, Seoguipo-si

A total of 298 mosquitoes were collected in Wimi-ri, Seoguipo-si, Jeju-do, comrpsing four genera, five species. Cx pipiens was most frequently caught (77.9%), followed by Och togoi (15.8%), Cx triraenior (5.4%), Ar subalbatus (0.7%), and Ae albopirtus (0.3%). No filariasis DNA was detected in Och togoi and An (Hyicanus) group (Table 6).

4. Discussion

In the survey, the ratio of Och togoi was highest in Maemul Island, Bogil Island, and Heuksan Island, while Cx pipiens was most frequently caught in Geomun Island, Namone-ri, and Wimi-ri in Jeju-do. This trend remained consistent in the survey between 2002 and 2005 by Cheun et al [19], and Cx pipiens also found out

Table 4.

Seasonal variations of mosquitoes collected in mosquito trap in Maejuk-ri (latitude 34°38'N, longitude 128°34'E), Maemul lsland, Tongyoung-si, Gyeongsangnam-do in 2009

Month	Ae albopirtus	Arm sabalbatus	Cx Bitaenior	Cx ipiens	Cx tritaenior	Och koreicus	Och togoi	Mean no./trap
May		7					23	30
Jun		16		2		2	175	195
Jul	2	47		2			278	329
Aug		34		4			297	335
Sep	2	38		10	3		220	273
Oct		3	1	7	3		37	51
Total (%)	4 (0.3)	145 (12)	1 (0.1)	25 (2.1)	6 (0.5)	2 (0.2)	1030 (84.9)	1,213

Numbers in parenthesis represent data on male mosquitoes.

to be the dominant species in Geomun Island and Jejudo. According to Ree [20], there are nine genera of mosquitoes found in Korea: Anopheles, Culex, Ochletotatus (formerly known as Aedes), Armigeres, Mansonia, Heizmannia, Tripteroides, Culisera, and Tozorhynchites. Among them, Och togoi was recognized as the main vector mosquito of B malayi in Jeju-do, Korea [8,21], whereas An sinensis sensu stricto, associated with rice paddies, is found in inland areas [9,14].

According to Lee [22], two larvae from mosquitoes were found to be infected with B malayi in 464 Aedes togoi collected in Jeju-do; after this, several researchers found that the natural infection rate of Aedes togoi was between 1.4% and 10.7%, and Och togoi was a vector of Malayan filariasis in Jeju-do [23-25]. However, Ae koreicus and Cu pipens were reported to have poor susceptibility as a vector of filariasis [26]. Furthermore, Kim et al [10] also collected 4351 An sinesis in Youngju-si, Chungcheongbuk-do, inland areas, and found that 14 mosquitoes (0.3%) were infected with B malayi. Therefore, these studies showed that both Och togoi and An sinensis had susceptibility for lymphatic filariasis [10]. In this study, An (Hyrcanus) group and Och togoi collected in these past endemic areas, however, were not infected with B malayi, and this fact is proof that these areas are free of lymphatic filariasis.

Next, mosquito larvae are susceptible to salinity and temperature, and their numbers increase after the

Table 5.

Seasonal variations of mosquitoes collected in mosquito trap in Namone-ri (latitude 33°16'N longitude 126°39'E), Seoguipo-si, Jeju-do in 2009

Month	Cx mineficus	Cx pipiens	Cx tritaenior	Och togoi	Mean no./trap
Jun		9		9	18
Jul		56	2	7	65
Aug		52		4	56
Sep		53	10	9	72
Oct	1	32		3	36
Total (%)	1 (0.4)	202 (81.8)	12 (4.9)	32 (13)	247

Numbers in parenthesis represent data on male mosquitoes.

Table 6.

Seasonal variations of mosquitoes collected in mosquito trap in Wimi-ri (latitude 33°16'N, longitude 126°39'E), Seoguipo-si, Jeju-do in 2009

Month	Ae albopirtus	Arm sabalbatus	Cx pipiens	Cx tritaenior	Och togoi	Mean no./trap
May			1	6		7
Jun		1	45	9	7	62
Jul	1		35		11	47
Aug			32		13	45
Sep		1	69		11	81
Oct			50	1	5	56
Total (%)	1 (0.3)	2 (0.7)	232 (77.9)	16 (5.4)	47 (15.8)	298

Numbers in parenthesis represent data on male mosquitoes.

summer monsoon season [19]. As the survey results show, the number began to climb up in June in all survey areas, stayed high July through September, and started to fall in October. According to Nakamura (1988), the number of Och togoi larvae begins to increase slowly when winter is over, and peaks in early May. The number falls in summer as the air and water temperature rises with strong sunlight. However, when rainfall increases in June through September, the number increases again due to lower salinity in rock pools [27]. This means that seasonal condition and regional characteristics have a direct influence on the number of mosquito larvae. Compared to a previous study [21], the regional decrease or increase in the number of collected mosquitoes was affected by these conditions. However, this study confirmed that Och togoi and An (Hyrcanus) group are the dominant species in survey areas.

This is the first study conducted in Korea that investigated filariasis DNA from vector mosquitoes in six remote island areas in Korea, and the negative results indicate that filariasis has been eliminated in the country. In particular, the costal island areas in Jeollanam-do showed a high infection rate until the early 2000s [28], but government, academic, and local clinics have been exerting efforts continuously to bring about its elimination. As the country’s economic growth accelerated from the late 1980s, peoples’ quality of living improved. They had better access to medical treatment, and various mosquito-repelling chemicals and equipment were developed such as mosquito nets. All these factors substantially reduced human contact with mosquitoes [29].

Finally, the survey examined the number of mosquitoes, including vector species for filariasis, in island areas of Korea, and confirmed the negative result in DNA detection. However, filariasis has a latent period of 4–10 years, and there is a possibility of its emergence or reemergence through travel or trade as more people visit areas in the West Pacific, Southeast Asia, and Africa, where filariasis is still active. For this reason, it is important to continue quarantine inspection and monitoring of vector mosquitoes.

Acknowledgements

We thank our colleagues at the Province and City Bureau of Health Center and the Research Institute of Health and Environment for their devoted support and efforts. This work was supported by a grant from Korea National Institute of Health (NIH-091-4800-4845-300), National Research and Development Program, Ministry of Health and Welfare, the Republic of Korea.

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Figure & Data

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Citations

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