Background
Plasmodium vivax malaria has re-emerged in many malaria-endemic areas, where the disease was believed to have been eradicated [
1]. In South Korea, it was assumed that malaria had been eradicated, since no indigenous case was reported after 1978. However, following the detection of a case in 1993, the annual incidence of malaria increased dramatically and reached a peak of 4,142 cases in 2000 [
2‐
5]. In the meantime, malaria cases in North Korea increased from 1,085 in 1998 to 296,540 in 2001 [
6]. As a result, malaria has gained renewed attention as a public health problem throughout the Korean Peninsula.
So far, eight anopheline mosquitoes have been reported from South Korea. Six of these species belong to the Hyrcanus group:
Anopheles sinensis,
Anopheles lesteri,
Anopheles pullus,
Anopheles sineroides,
Anopheles kleini and
Anopheles belenrae [
7‐
10]. Two other non-Hyrcanus group species are
Anopheles koreicus and
Anopheles lindesayi japonicus [
7].
Since vivax malaria has been recognized as an endemic disease particular to countries in the Far East Asia, including Korea, efforts to find the main vector(s) have continuously proceeded from Korea, Japan and China [
7,
11‐
13]. However, there is currently no available report regarding the vector susceptibility of the two non-Hyrcanus species and
An. sineroides, because these species are very rare in Korea, Japan and China and, are thus considered medically less important [
7,
14,
15]. Although there is some published literature on the two newly reported species,
An. belenrae and
An. kleini [
9], the biting behaviour, larval habitats, and medical importance of these species are still unknown [
16]. The results of older studies on the vector abilities of
An. lesteri,
An. sinensis and
An. pullus are controversial.
Anopheles sinensis has long been considered a primary and strong vector in Korea and Japan [
11,
12,
15,
17], while in countries like China and Thailand, this mosquito is treated as a refractory vector with weak transmission capability [
13,
18,
19].
Although
An. lesteri has been considered a minor and weak vector in Korea and Japan, studies in China showed that it was an important vector [
20]. Conflicting reports on the vector ability of
An. pullus may also be found [
12,
4], with some suggesting that it is a malaria vector whereas others indicate otherwise [
21]. Thus, the malarial susceptibility of these vectors remains to be clarified.
In this study, three species of the Hyrcanus group, An. lesteri, An. sinensis and An. pullus were experimentally infected with a Korean isolate of P. vivax and the malarial susceptibility of these species was analysed based on their ability to develop oocysts in the midgut and sporozoites in salivary glands. To substantiate the findings of the above experiments and to further understand and verify the existing differences in vector abilities of two geographically distant strains of An. sinensis from Korea and Thailand, experimental infections were conducted with a Thai isolate, and the ability of both these strains to develop oocysts and sporozoites was determined.
Methods
Colonization of mosquitoes
Three mosquito lines of the Korean
Anopheles raised in the laboratory were
An. lesteri,
An. sinensis and
An. pullus. Among these,
An. sinensis and
An. pullu s were collected from Paju City, Gyeonggi-do Province, while
An. lesteri was collected from So-Rae District, Incheon City in South Korea. The Thai strain of
An. sinensis was collected from Mae Tang District, Chiang Mai Province, Thailand.
Anopheles cracens was in a free-mating colony established for more than two decades in the insectariums of the Department of Parasitology, Chiang Mai University, Thailand and was used as a control, since it is known to be highly susceptible to
P. vivax [
22]. These colonies were reared using the method described by Park
et al [
23] in an insectary room at 27 ± 2°C, 70–80% relative humidity with 12:12 hr light and dark photoperiods adjusted by fluorescent lighting.
Infected blood
Blood containing gametocytes of Korean P. vivax was supplied by the National Institute of Health (KNIH), from a patient seeking treatment at Gimpo, Gyeonggi-do province. The Thai strain of P. vivax was obtained from malaria patient infected in Mae Tang District of Chiang Mai Province. Informed consent was obtained from the patients before collection and the study protocols were approved by Internal Review Board of Korea National Institute of Health and Thailand Office for Vector Borne Diseases Control, Department of Communicable Diseases Control, Ministry of Public Health. Giemsa staining of the blood film was performed and gametocytes were counted. The gametocyte density of the Korean isolate of P. vivax was 36 gametocytes/200 White Blood Cells (WBC) while that of the Thai strain was 26 gametocytes/200 WBC.
Infection of mosquitoes with P. vivax gametocytes
Three to five days old females were used for infection. To enhance their willingness to feed, the mosquitoes were starved for 12 hours prior to infection and were transferred to paper cups of size 8.5 cm in diameter and 11 cm in depth (about 50 females per cup). The mosquitoes were fed on infected blood containing gametocytes through an artificial membrane feeding technique described by Chomcharn
et al [
24]. The mosquitoes were allowed to feed for one hour.
Unfed mosquitoes were removed and the fully engorged females were carefully handled and were kept in the insectary. Mosquitoes were then fed on cotton patches, which were soaked in 5% sucrose solution and changed every day, until the mosquitoes were dissected. During infection, three lines of the Korean mosquito: An. lesteri, An. sinensis and An. pullus were infected with Korean isolate of P. vivax, while strains of An. sinensis from Thailand and Korea together with An. cracens were infected with Thai isolate of P. vivax.
Eight and 14 days after feeding, mosquitoes were dissected to detect oocysts in the midgut and sporozoites in the salivary glands respectively. Counting of oocysts in mosquitoes was followed by examining wet mounts of the midgut stained in 0.1% mercurochrome and freely moving sporozoites were carefully detected from salivary glands placed in a drop of phosphate buffered saline (PBS, pH 7.2).
To explore differences in densities of the salivary gland sporozoites between
An. lesteri and
An. sinensis, two methods were used: 1) for
An. lesteri, micrographs were taken at 100× magnification using a camera (Motic Cam 2000) mounted on a compound microscope (Leica DM 2500) and counting of sporozoites was performed within a captured micrograph, the corresponding area of which was approximately 750 μm × 560 μm; 2) sporozoites were counted from whole salivary glands from
An. sinensis and were compared to the sporozoite loads within a single microscopic field of
An. lesteri. The above counting and comparative procedures were developed because in highly infected
An. lesteri, the direct counting of several hundreds of moving sporozoites while observing through the microscope was difficult. It was sometimes impossible to count all of them from salivary glands while in
An. sinensis any such possibilities were less and few numbers of sporozoites were directly counted by changing fields. Depending on counting, mosquitoes were graded as 1+ (1–10 sporozoites), 2+ (11–100 sporozoites), 3+ (101–1,000 sporozoites) and 4+ (above 1,000 sporozoites) [
25]. The total number of +'s recorded for all infected mosquitoes was gland indexes in terms of sporozoites load.
Discussion
The abundance of
An. sinensis in the Orient led to early suspicions of its role as a vector, but questions as to its role have continued for several decades [
13,
15,
18]. The first successful study on experimental malarial transmission in
An. sinensis was conducted by Tsuzuki in Japan, in 1902, which Kamimura suggested later on was actually
An. lesteri [
7]. Subsequently, Otsuru and Ohmori [
15] stated that
An. sinensis had a similar ability to develop malaria as
An. lesteri in Japan.
Ho
et al [
13] showed that
An. lesteri, besides being the primary vector of filarial parasites, was also the primary vector of malarial parasites in the hilly regions of the Yangtze valley (South China) and also pointed out that
An. sinensis, due to its zoophilic behaviour, is an inefficient vector responsible for maintaining a low malaria endemicity in the broad flat rice plains of south China. Similarly, Harrison [
26] opined that
An. lesteri might well be a more efficient vector than
An. sinensis. Lately, Liu [
20] recorded a high number of infections in
Anopheles anthropophagus (synonymized as
An. lesteri) [
27] from experimental feeding with
P. falciparum gametocytes and also reported the natural infection rates of
An. lesteri to be higher than
An. sinensis in China. With such findings and along with other essential parameters (human-biting rate, human blood index, vectorial capacity and entomological inoculation rate),
An. lesteri was considered to be 20 times more efficient as a malaria vector than
An. sinensis in malaria infected regions in China [
20,
28]. In South Korea however, the first natural infections in
An. sinensis were reported in 1962 [
11]. In a similar manner, Hong [
12] also reported a very low number of sporozoite positive
An. sinensis followed by
An. pullus (previously called
Anopheles yatshushiroensis). These early studies supported the primary and secondary vector status of
An. sinensis and
An. pullus.
In recent years, much of the understanding of vector susceptibilities of Korean
Anopheles was based on Enzyme-Linked Immunosorbent Assay (ELISA). Most of these studies indicated
An. sinensis to be a vector [
21,
29,
30], while recently, Lee
et al [
4] reported
An. kleini and
An. pullus to be stronger vectors than
An. sinensis. Because Circumsporozoite Protein (CSP) detected by ELISA can equally be detected from developing oocysts, and sporozoites present in haemocoel, positive results from the test do not always indicate that the salivary glands are infected with sporozoites [
31]. Thus, immunological evidence supporting the presence of CSP may indicate that the mosquito is a potential vector of malaria, but it is not proof that the sporozoites are located in the salivary glands and can be transmitted to a vertebrate host by a mosquito bite.
In this study, three Korean species of Hyrcanus group (
An. lesteri,
An. sinensis and
An. pullus) were experimentally infected with an indigenous Korean isolate of
P. vivax. After eight and 14 days post-infection, examination of midguts showed the presence of high number of oocysts in all three species. However, there were differences in their innate ability to develop sporozoites in the salivary glands. Sporozoites in salivary glands were detected from
An. lesteri and
An. sinensis but not from
An. pullus. Though, sporozoites were detected from
An. sinensis, they were very few as compared to
An. lesteri. The maximum number of sporozoites in a salivary gland of
An. sinensis was 14, which corresponded to the findings of Rongsriyam
et al [
19]. Assuming that two salivary glands have an equal number of sporozoites, fewer than 30 sporozoites are likely to develop in a pair of salivary glands of the above-mentioned mosquito. Earlier Ito
et al [
32] reported that mean densities of the sporozoites below 400 were not sufficient enough to initiate infections in mice. In such contest, low numbers of sporozoites detected from
An. sinensis can be assumed to play less important role in initiating infections when compared to
An. lesteri.
Because sporogony in
P. vivax at 25°C is completed within 9 days [
33], fourteen days time in this study, after which salivary glands were examined, should have been sufficient for sporozoites to reach to salivary glands. But compared to
An. lesteri, very few number of
An. sinensis contained salivary gland sporozoites however no sporozoites were detected in
An. pullus. This suggests that developmental transitions couldn't proceed from oocysts to sporozoites formation in
An. sinensis and
An. pullus. Beier
et al [
33] described that inhibition in transitions from oocysts to sporozoites might be caused due to different mechanisms like oocysts failing to produce sporozoites, sporozoites failing to navigate successfully to the salivary glands, invading salivary glands or surviving in the salivary glands.
In addition to demonstrating sporozoites in salivary glands following laboratory infection, it is necessary to consider the natural survival rates of malaria vectors. At present, there are no connected reports with the matter in An. lesteri. Therefore, future studies regarding survival rate of An. lesteri in wild population can be more supporting evidence for this study. In Korea, the present study is the first of its kind, comparing the malarial susceptibilities of these members through successful malaria development within lab-raised clean colonies. The results are well congruent with Chinese and Thailand reports. So, the outcomes of this study have significant bearings within entire temperate regions where these species are abundant.
Conclusion
Under laboratory conditions, salivary gland sporozoites were developed in An. lesteri, as readily as in the well-recognized vector. Big differences were seen in the rate and densities of sporozoites between An. lesteri and An. sinensis, whereas sporozoites were not detected from salivary glands of An. pullus even after 14 days of oocysts development. Also, geographically distant strains of An. sinensis from Korea and Thailand were similar in their ability to support malaria development. Therefore, An. lesteri is highly susceptible to P. vivax malaria as compared to An. sinensis and An. pullus.
Thus, An. lesteri is a potential malaria vector and its presence may be described as an under-rated public health threat. However, comparative susceptibility of the remaining members of the Hyrcanus group will be important in future to understand their role in malaria epidemiology in Korea.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MGS designed the present study. Prior to this experimental work, WC facilitated DJ and MHP for training on malaria susceptibilities and improved lab colonization of Anopheles mosquitoes. DJ and MHP conducted all experimental studies under the supervision of MGS and WC. Malaria-infected blood was obtained from WC, WS, TSK and JYK. DJ drafted the manuscript with MHP. All the authors read the manuscript.