Characteristics of potential breeding sites for Anopheles in the study sites
Malaria is continuously prevalent in Korea, showing an average of 500 patient cases annually, and COVID-19 disrupted malaria services, leading to a significant increase in cases and deaths worldwide [18]. Interestingly, the number of malaria cases in Korea decreased below 274 during the COVID-19 pandemic. However, subsequently, it rose more than 388 indigenous cases according to the Korea Disease Control and Prevention Agency in 2022 [19] (Fig. 1A)
Gyeonggi-do province near the DMZ had the highest malaria patient incidence, and cases significantly increased in 2022 (Fig. 1B). According to the Korea Disease Control and Prevention Agency, the malaria incidence rate per 100,000 population was more than 2% in Gyeonggi-do province in 2022; thus, we explored five village districts (WL, Wollong-myeon; PJ, Paju-eup; MS, Munsan-eup; HS, Haseong-myeon; WG, Wolgot-myeon) near the DMZ area (Fig. 1C).
Figure 1D shows the location on satellite image data of adults and larvae of mosquitoes recorded in this study. According to visualization in Google Earth, the collection sites of the two regions in Goyang and Gimpo city were geographically different.The collection spots of Goyang city (WL, PJ, and MS) were chosen from primarily cultivated land with rice fields, while those of Gimpo city (HS and WG) were close to mountains and rice fields in a 1 km radius. Moreover, except WG, four villages have cattle sheds within a 1 km radius.
Differential Distribution Of Larvae And Adults Of Mosquitoes
Previous studies have demonstrated that An. sinensis larvae were commonly found in ground pools, plastic containers, and swamp and rice fields [9, 20].
Therefore, we searched the irrigation ditches and rice paddies in Goyang city from May 2022 because the current abundance of larvae of An. sinensis in DMZ areas was not well reported (Fig. 2A-F).
In the Goyang areas, seven species (An. belenrae, An. kleini, An. lesteri, An. lindesayi, An. pullus, An. sinensis, and An. sineroides) have been reported [21]. An. sinensis and An. lesteri populations peaked in August, whereas An. pullus peaked in May and decreased from June to October in Korea [22].
In our study, larvae and adults of Aedes and Culex were prevalent in Goyang city (Table 1).
Breeding sites of Aedes and Culex were easily noticed at plastic buckets at cultivated land in WL (Fig. 2A-C). This land was surrounded by more than 20 households and placed near a rice field within a 1 km radius. Aedes and Culexlarvae at Goyang cities were co-habituated in plastic buckets, bowls, trays, tile jars, and turbid plastic buckets in the WL district as previously reported [23]. However, no larvae and adults of Anopheles spp. were recorded in WL, PJ, and MS in our study. PJ and MS showed positive larvae of only Aedes and Culex in human dwellings (Fig. 2D-F). Rice filed or stream in PJ and MS showed no Anopheles larvae.
Gimpo city was reported to be a popular place with a high abundance of larvae of An. sinensis in Korea, such as Goyang city [21]. Therefore, we further investigated the two places of Gimpo from the end of July to early October. While Anopheline mosquito larvae were not found in rice fields or ditches near the village of Goyang city, Anopheles larvae were temporarily found in the HS village on July 19th (Fig. 2G). We explored other places near HS and WG was chosen for further investigation.
On the west side of WG village was a rice field, and on the east side was a forest stream (Fig. 2H and I). Anopheles larvae lie parallel to the water surface to breathe air through spiracular openings (Fig. 2J).
Adults of Anopheles are morphologically distinguished from Aedes and Culex by the presence of thick palpi and proboscis in males and females (Fig. 2K and L).
A forest stream of permanent breeding site was secluded by forest trees and adults were captured in village within 100 m distance (Fig. 2M).
Table 1
Distribution of mosquitoes larvae and adults at Goyang city in 2022
Area
|
WL
|
PJ
|
MS
|
Date
|
May 27th
|
June 24th
|
July 8th
|
July 29th
|
August 18–19th
|
September 7th
|
July 8th
|
July 8th
|
Temperature (℃)
|
19.5–22.2
|
24.9–25.0
|
25.8–26.6
|
31.1–32.3
|
26.1
|
26.9
|
26.7
|
21.1–24.1
|
Humidity (%)
|
54–62
|
73–75
|
82–85
|
62–75
|
74–75
|
32
|
82
|
92–99
|
Total number of An. mosquitoes spp. (larvae)
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Total number of An. mosquitoes spp. (Adult)
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Total number of Aedes and Culex (Adult) in trap among five traps
|
10
|
20
|
-
|
-
|
6
|
-
|
-
|
-
|
Total number of Aedes and Culex (Larvae)
|
47
|
> 100
|
> 100
|
> 100
|
> 200
|
> 50
|
> 50
|
> 50
|
WL, Wollong-myeon; PJ, Paju-eup; MS, Munsan-eup |
Table 2
Distribution of mosquitoes larvae and adults at Gimpo city in 2022
Area
|
HS
|
WG
|
Date
|
July 29th
|
August 4th
|
August 4–5th
|
August 10–11th
|
August 18–19th
|
August 24–25th
|
August 31st–September 1st
|
September 7th
|
September 20–21st
|
September 28–29th
|
October 6–7th
|
Time
|
22:00–23:30
|
22:00–24:00
|
22:00– 24:00
|
22:00–08:30
|
22:00–07:30
|
20:00–09:00
|
19:00–06:00
|
15:00
|
20:00–07:00
|
18:30–09:00
|
21:30–08:30
|
Temperature (℃)
|
26.6–27.5
|
25.0–25.8
|
25.0–26.5
|
20.7–23.6
|
21.1–24.1
|
19.5–23.1
|
17.7–23.0
|
27.0
|
9.6–18.0
|
16.3–20.3
|
10.7–14.9
|
Humidity (%)
|
91–94
|
98–99
|
98–99
|
93–99
|
92–99
|
87–99
|
83–99
|
42
|
57–94
|
86–99
|
78–91
|
Total Trap number
|
3
|
3
|
2
|
5
|
5
|
5
|
5
|
-
|
5
|
5
|
3
|
Positive trap number
|
2
|
0
|
2
|
5
|
4
|
5
|
3
|
-
|
3
|
2
|
2
|
The total number of captured An. mosquitoes spp. (Adult)
|
n = 2
|
n = 0
|
n = 5
|
n = 22
|
n = 6
|
n = 12
|
n = 5
|
-
|
n = 6
|
n = 2
|
n = 2
|
PCR bands
/Captured mosquito
|
2/2
|
0/0
|
4/5
|
15/22
|
0/6
|
3/12
|
2/5
|
|
5/6
|
2/2
|
2/2
|
Total number of An. mosquitoes spp. larvae
|
11
|
0
|
-
|
-
|
-
|
> 50
|
> 50
|
> 50
|
> 50
|
> 50
|
< 20
|
Total number of An. mosquitoes spp. larvae outside stream
|
-
|
-
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Total number of Aedes and Culex adults in the trap
|
> 10
|
> 10
|
> 10
|
> 10
|
> 50
|
> 10
|
> 10
|
-
|
< 10
|
< 10
|
< 10
|
Total number of Aedes and Culex larvae
|
> 50
|
> 50
|
-
|
-
|
-
|
> 10
|
< 10
|
> 10
|
< 10
|
< 10
|
< 10
|
Table 3
Distribution of Anopheles adult at Gimpo city in 2022
Area
|
HS
|
WG
|
Date
|
July 29th
|
August 4th
|
August 4–5th
|
August 10–11th
|
August 18–19th
|
August 24–25th
|
August 31st–September 1st
|
September 7th
|
September 20–21st
|
September 28–29th
|
October 6–7th
|
An. sinensis
|
1
|
0
|
3
|
13
|
|
2
|
2
|
-
|
2
|
2
|
1
|
An. pullus
|
1
|
0
|
1
|
|
|
|
|
-
|
|
|
|
An. sineroides
|
|
0
|
|
2
|
|
|
|
-
|
|
|
|
An. belenrae
|
|
0
|
|
|
|
|
|
-
|
2
|
|
1
|
An. lindesayi
|
|
0
|
|
|
|
1
|
|
-
|
1
|
|
|
HS, Haseong-myeon; WG, Wolgot-myeon
Distribution of mosquitoes in Goyang and Gimpo city
Recently, the earliest appearance of Anopheles was reported in 2020, showing the highest peak in June and August [1].
The larval sampling was conducted using the standard dipping method at 10 times dipping with a plastic container (30 cm ⅹ 50 cm ⅹ 20 cm) as previously described [9].
Table 1 shows the villages with only Aedes and Culex larvae but the absence of the Anopheline in Goyang city. The temporal water sources of land and house dwelling continually maintained the high abundance of larvae and pupae of Culex and Aedes consistently.
Table 2 shows the characteristics of potential breeding sites of Anopheles larvae in the two study sites in Gimpo city. HS showed the transient larvae and adults of Anopheles on July 29th rather than on August 4th. In contrast, WG showed larvae and adults of Anopheles consistently from August to October, indicating that WG is a permanent place of the breeding site.
The results imply that Anopheles larvae were spatially distributed in different aquatic habitats in Gimpo city.
In 2022, it was odd that the heavy rainfall of July was extended to October compared to the previous years (Fig. S1). Therefore, it might cause an overall reduction in the positive rate of mosquito adults captured in the trap in the field. Lasting raining season would be one of the main reasons to obtain fewer mosquitoes captured in trap because the temperatures were not variable from July to October during the last 4 years.
WG stream maintained more than 50 Anopheles larvae from August 24th to September 28th and decreased to less than 20 larvae in early October.
GenBank Accession numbers of raw sequences of inter-transcribed spacer region 2 (ITS2) are presented in Table S1. Overall, An. sinensis was the most prevalent spp. showing that An. sinensis, An. pullus, An. sineroides, An. belenrae, and An. lidesayi accounted for 41.67% (25/60), 1.67% (1/60), 3.33% (2/60), 5.00% (3/60), 3.33% (2/60) (each mosquito spp./total Anopheles adult) at WG stream, respectively (Table 3).
The highest prevalence of Anopheles mosquitoes collected from May to early November 2020 was reported with An. kleini (31.4%), followed by An. pullus (25.5%), An. sineroides (23.9), and An. sinensis (10.2%) while the other four species only accounted for 9.1% (147/1622) collected in/near the DMZ [1], it is a different result from this study.
All Anopheles adults captured in this study showed the negative result of P. vivax in RT-PCR (Fig. S2).
As shown in Fig. S3, the two cities shared a similar weather characterization of the same pattern of temperature and precipitation according to Korea Meteorological Administration, National Climate Data Center (Republic of Korea).
Factors associated with Anopheles larval density
Habitats of Anopheles larvae were widely identified [24], and several known positive parameters of An. sinensis larvae were reported as water bodies in the village, such as water depth, predator, pH, and chemical oxygen demand [9].
Although it has been reported that the larvae of An. sinensis can be reared with yeast [12], we found that An. sinensis was different with the breeding condition of those of Aedes(Fig. S4).
Therefore, Anopheles larvae were kept in plastic trays containing original stream water and were maintained in laboratory conditions more than 20 days, and their motility was improved in vitro culture condition (Video S1).
As larvae were found in fresh water in a forest stream, we hypothesized that the dissolved oxygen (DO) was different in that of forest stream because Anopheles larvae were not found in any turbid place at the village within a 100 m distance.
We measured the DO level of forest sream of WG, which was significantly different from tap water.
As shown in Fig. 3A, DO in WG was significantly different from that of tap water (P < 0.001). Therefore, the effect of the increased oxygen on Anopheles larvae growth was further investigated in laboratory conditions.
We installed a different number of air diffusers in the plastic container (17 cm ⅹ 10 cm ⅹ 11 cm) to aid aeration with an increased oxygen supply.
All experiments were conducted at 27 ± 2°C and 75–85% humidity under a 14:10 light/dark photoperiod.
Tap and WG forest water showed 8.45 ± 0.11 and 9.7 ± 0.17 (mean ± SD) mg/L of DO, respectively. L1-L2 larvae (n = 25) (length of larvae body < 0.5 cm) were cultured in a forest stream, and after the 3rd day since culture, fresh WG stream water was added to the water (1:1) and the DO, survived larvae, and pupae were monitored after development.
As found in Fig. 3B, the DO amounts were different with a different number of air diffusers, and the estimation plot showed a significant difference in DO between single and three air diffusers (Fig. 3C) (P < 0.05).
The larvae survival rate was significantly higher in the three air diffuser conditions than that of the single diffuser, maintaining more than 20% survival at 24 days, while the single diffuser showed all larvae death at 18 days (P < 0.01)(Fig. 3D).
Two pupae at three air diffusers were developed, while one was generated in a single air diffuser. It indicates that the concentration of DO might influence larvae growth and pupae development.