Distribution Of Anopheles Mosquitoes , Potential Vectors, Insecticide Susceptability And Bio-Efficacy Of Llins Nets In Different States And Regions In Myanmar
Mya MM, Thaung S, Myint YY, Mu TN and Maung YNM
Published on: 2022-04-09
Abstract
Vector bionomic potential vector, sibling species, insecticide subceptibility and efficacy of LLINs nets were carried out in Indaw, Tangoo, Shwe Kyin, Beelin, Than Byu Zayat, Kamamaung, Hpa-an and Pyin Oo Lwin Townships in Myanmar from June 2018 to May 2020. Adult mosquitoes were collected using cattle baited net, indoor and outdoor light traps methods and larval survey was conducted.Standard WHO insecticide susceptibility was employed using field collected specimens as well as vector incrimination and polytene chromosome study were done for sporozoite and sibling species identification. 80 used Long Lasting Insecticidal Nets (LLINs) 10 each from eight malaria endemic areas of States and Regions of Myanmar were assessed for their bio-efficacy against Anopheles mosquitoes. Insecticide susceptibility and bio-efficacy test were done according to WHO testing method. Results revealed that a total of 6172 mosquitoes of 14 Anopheles species including main vector and secondary vectors were collected in high density in Than Pyu Zayat and Beelin in Mon State, Kamamaung and Hpa-an in Kayin State, Shwe Kyin and Tangoo in Bago Region, Pyin Oo Lwin in Mandalay Region and Inn Daw in Sagaing Region. Three Sibling species as An minimus A, An. dirus D and An. culicifacies B were observed and pick biting time was found 10:00 pm for An. dirus and 11:00 pm for An. minimus in study areas. All collected mosquitoes were found susceptible to WHO recommended insecticides. Bio-efficacy of PermaNet 2.0, DAWA TANA, Yorkool, BASF, Net protect and Yahe LLINs nets were found to be 90 to100% protection from vector Anopheles mosquitoes. The study suggested that there is need to distribute a large number of effective LLINs nets in malaria endemic areas as well as the replacement of expired, less effective and very old LLINs nets with a new one is also necessary. LLINs nets are very effective Anopheles mosquitoes control tools and it can reduce the human-vector contact effectively. Moreover, research on bio-efficacy evaluations of the LLINs nets is required regularly to maintain and control the transmission of malaria through Anopheles mosquitoes.
Keywords
Potential vector; Bionomics; An.minimus; An. dirus, Bio efficacy; Sibling speciesIntroduction
Previously Malaria is one of the major public health problems in Myanmar and it is still a main contributor to the morbidity and mortality in hard to reach areas and border line areas in the country. In malaria prevention and control, use of insecticide treated bed nets and application of focal residual spray at selected areas are important part of the programme in endemic regions. An. dirus and An. minimus are major vectors of malaria in forested and forested foot hill areas of the country and An. annularis is a local vector of malaria in Rakhine State. An. sundaicus and An. subpictus are abundantly found in coastal areas of Rakhine, Mon and Tanintharyi Regions. The transmission of malaria is largely influenced by the abundance, survival ecology, topography and bionomics of the vector mosquitoes [1,2]. The main vector control tools, long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) at focal areas can be very effective in reducing malaria transmission. They are more effective against vectors which bite indoors (endophagic), late in the night (nocturnal) and which rest indoors after feeding of blood meal (endophilic) [3]. These characteristics are observed for the primary malaria vector like An. minimus and are contributing to the success of the malaria control programmes in this region. However, it might be possible that after widespread and long-term insecticide exposure, the feeding and resting behaviour of vectors may become much more variable. Furthermore, vector mosquitoes may develop tolerance and leading to Pyrethroid resistance in endemic areas [4]. At the same time, residual effectiveness of the Insecticide treated bed net (ITN), long lasting Insecticidal nets (LLINS) and IRS should be monitored periodically for judicious use of insecticides and sustainable vector control [5]. The operational criterion of resistance has usually been taken as the survival of 20% or more of the individuals tested at the currently known diagnostic concentrations using WHO test kits in field [6]. Therefore, updating and better understanding of the bionomics of vector mosquitoes are required together with monitoring of the insecticide susceptibility status and measuring of the residual effectiveness of insecticide treated surfaces at malaria endemic regions. It is essential to detect efficacy of currently used insecticides in the country and available information will be useful to complement the control programme. Present study planned to investigate the bionomics, potential vectors, susceptibility of malaria vectors and evaluate the efficacy of insecticide treated long lasting nets for strengthening of control strategies.
Rationale
Inn Daw Township Sagaing Region, Tangoo and Shwe Kyin Township in Bago Region, Than Byu Zayat and Beelin Township Mon State Kamamaung and Hpa-an Township Kayin State and Pyin Oo Lwin Township Mandalay Regions are festered hilly areas and most of the people live in hilly areas where main vector An. dirus and An. minimus are abundantly available [7]. 60 to100 households and 2000 to 20000 population are available in each area. Many creeks traverse the mainland with tributaries of varying lengths. The topography of the areas are abounded in evergreen forests, rice fields and rubber plants, rubber plantations provide a living for many and the locals also maintain a remarkable range of traditional cottage industries. The climate is tropical (4° to 30°C temperature) and humid (mean relative humidity of 78.5 to 90%) with abundant rainfall, supporting a luxuriant and rich vegetation. All state and regions are a malaria endemic area and primary and secondary vectors are abundantly present in this area [1] and larvae of main vector An. dirus are abundantly found in domestic water wells, water pools and rock pools [8]. According to the ecological changes, distribution of malaria morbidity patterns and results from the micro-stratification, the high risk areas for the malaria was about 38.90% in the 1990 was reduced to 17.00% in 2013. And free areas in the country were increased from 8.60% in 1990 to 37% in 2013 [9]. Large numbers of native and migrant workers are working in gold mining, rubber plantation and handmade factory’s and contracted sites and they have needed to protect from vector borne diseases. And also there is a lack of data and information about the potential vectors, susceptibility, bio-efficacy of malaria in selected areas in State and Regions of Myanmar. Therefore, an attempt has been made to determine the vector bionomics, potential vectors, insecticide susceptibility and bio- efficacy of primary and secondary vectors of malaria in these areas.
Materials and Methods
Study Areas: The study was conducted in Inn Daw Township Sagaing Region, Tangoo and Shwe Kyin Township in Bago Region, Than Byu Zayat and Beelin Township Mon State Kamamaung and Hpa-an Township Kayin State and Pyin Oo Lwin Township Mandalay Regions.
Study Period: Study period was 2 years, started from June 2018 and ended in May 2020.
Data Collection Procedures
Mosquito Collection: Fixed mosquito catching stations were chosen in both selected villages. Indoor, outdoor light traps and animal bait K Net (330 x 330 x180cm) big mosquito net trap catches were conducted seasonally. All catching was done in fixed stations throughout the study period. Animal bite, both indoors and outdoors light traps catching were done with WHO sucking tubes from 18:00 to 06:00 hours of the next day for 7days.
Larval Surveys: For identification of breeding sites larval survey was conducted in and around three kilo-meters away from the study villages. Dam, canal and irrigation, domestic wells, stream/creeks and all different types of water holding places were examined for larvae detection by 3 Dips /water holding place [10] (WHO 1975). In the rainy season all water pockets, coconut shells, discarded tins and utensils bamboo stumps including foot print of animals were examined. The captured larvae and pupae were put in labeled plastic bags and brought back to the laboratory for species identification and colonization.
Identification of Adult Anopheles Mosquitoes
Collected adult Anopheles mosquitoes and adult emerged from larva survey were identified by species according to different authors [11,12,13].
Insecticide Susceptibility Test
Insecticide Susceptibility Tests (WHO Test Kit): Adult female Anopheles mosquitoes collected from the entomological survey was tested for measurement of insecticide susceptibility level using WHO test kits [14] and standard procedures. The efficacy of insecticides which are commonly used for malaria vector control in malaria endemic areas namely as DDT4%, Permethrin 0.75%, Cyfluthrin0.15% and Deltamethrin 0.05% were measured.
Procedure
10 fields collected Anopheles mosquitoes were introduced in WHO insecticide impregnated paper attached plastic tube (WHO test kit) [14] by sucking tube and exposed for 1 hour. After one hour of exposing the mosquitoes were then removed from the plastic tubes and placed in clean plastic tubes without paper with 10% glucose soaked cotton and moisture were maintained by water soak dump towel. Percentage of knockdown was measured after 60 min exposure and effective mortality was assessed after 24 hours’ exposure. Two replicates testing were done to confirm the susceptibility of mosquitoes. (10mosquitoes x 5insecticides (4test+1control) x 2 replicates =100 mosquitoes).
Bio Efficacy of Long Lasting Insecticide Nets (Llins)
Washed and unwashed in used for 1 to 2 years of 25 LLINs per site were collected randomly. Untreated standard polyester net was used as control. Bio-efficacy of long lasting insecticidal nets was evaluated according to standard method of WHO cone technique [15]. Insecticide susceptible strain of An. dirus colony from Medical Entomology Research Division was used before the bioassays as control. Two cones (one top, and one lateral) were placed horizontally on the nets and 5 each field collected adult female Anopheles mosquitoes (pool sample) were exposed to each cones for 3 minutes. After 3 minutes, mosquitoes were removed from the cones and placed in individual paper cups with assess to 10% glucose and humidity were maintained with water soaked gauze covered with on it. The percentage mortality was assessed 24 hours after exposure.
Incrimination of Vector
Head and thorax of vector mosquitoes were dissected for Plasmodium sporozoites. Enzyme Linked Immunosorbent assay (ELISA) test for circumsporozoite antigen detection was supplemented according to Wirtz et al., [16] for vector incrimination study.
Identification of Sibling Species
Preserved ovaries were processed in 50% propionic acid and were stained with 2% lacto-aceto-orcein stain according to the method Green and Hunt [17] for making polytene chromosome preparations. All collected samples were stored in a freezer at - 4ºC in laboratory till use. The chromosome complement of individual mosquitoes was examined under 40X and 100X magnification lens of compound light Olympus microscope for species diagnostic inversions were used for identification of the members of Anopheles minimus complex and An. culicifacies complex. Salivary gland polytene chromosome method was used for An. dirus complex.
Meteorological Data
Meteorological data as rainfall, Relative Humidity (RH) and Temperature (ºC) were obtained from TMEP Township weather stations.
Map of the Study Areas
Map of the study areas, adult Anopheles mosquito collection sites, (indoor, outdoor and animal bait) larva positive places and human settlement areas were drawn by using Global Positioning System GPS (GPSMap16 Garman, 18x-5HZ software interface application) method by expert person.
Data Analysis
Seasonal collected entomological data was analyzed and compared both study areas. Data entry, processing and analysis Seasonal collected entomological data was analyzed and compared both study areas. Data entry, processing and analysis were carried out using SPSS software applying Student ‘t’ test and ANOVA and Microsoft Excel (Analysis package) software and appropriate statistical analysis were applied as necessary.
Results
Distribution Of Anopheles Mosquitoes In Different States And Regions In Myanmar.
Figure 1: Map of Anopheles mosquitoes distribution in different areas in Myanmar.
(Figure 1) Shows detail of Anopheles mosquitoes distribution in different townships of States and Regions, main vector An. minimus was abuncuntly collected in all selected areas such as Inn Daw, Tangoo, Shwe Kyin, Beelin,Than Byu Zayat, Hpa-an, Kamamaung and Pyin Oo Lwin Townships. Main vectors An. dirus and An. minimus were observed in Kamamaung Township Kayin State. Anopheles minimus was collected in all study areas although the highest dendity of An. minimus (23%) was found in Pyin Oo Lwin Township followed by Tangoo (13%) and lowest density was found (4%) in Inn Daw Township Sagaing Region. Only 3 % of An. dirus was collected from Kamamaung Township Kayin State. Secondary vectors Such as An. maculatus, An, philippinensis, An. aconitus, An vagus, An.annularis and An.jamesii were collected in high percentage in all areas. An.annularis was high in Kamamaung(22%), Hpa-an(22%), Pyin oo Lwin(16%), and Beelin(16%) Townships and low density was observed (5%) in Inn Daw Township. An. philippinensis was found high density in Inn Daw (40%) followed by Kamamaung (18%) and lowest was observed (13%) in Hpa-an Township.
Assessment of the Insecticide Susceptibility Status of Anopheles Mosquitoes in Myanmar
Monitoring of insecticide susceptibility status in some Anopheles were conducted in Inn Daw Township, Sagaing Region, Beelin and then Byu Zayat Townships in Mon State, Tangoo and Shwe Kyin Township in Bago Region, Hpa-an and Kamamaung Township in Kayin State and Pyin Oo Lwin Township in Mandalay Region using WHO test kits. Batches of Anopheles were exposed to standard impregnated papers for standard exposure time of one hour. These studies were conducted to monitor the susceptibility of Anopheles mosquito to various insecticides in selected areas of Myanmar. There was no insecticide resistance detected in Inn Daw, Beelin, Than Byu Zayat Tangoo, Shwe Kyin, Kamamaung, Hpa-an and Pyin Oo Lwin Townships. Species of Mosquitoes and insecticide used were shown in below the table.
Table 1: Susceptibility status of Anopheles mosquitoes collected from different areas.
Township |
Anopheles species |
No. |
Tested Insecticides |
Remark |
In Daw Township, Sagaing Region |
An. minimus |
30 |
DDT 4%, Deltamethrin 0.05%, Permethrin 0.75% and Cyfluthrin 0.15% |
Susceptible |
An. vagus |
30 |
|
|
|
An. kochi |
30 |
|
|
|
An. Philippinensis |
30 |
|
|
|
An. annularis |
30 |
|
|
|
An. kawari |
30 |
|
|
|
An. barbirostris |
80 |
|
|
|
An. hyricanus |
30 |
|
|
|
An. tessellatus |
30 |
|
|
|
An. aconitus |
30 |
|
|
|
An. jamesii |
30 |
|
|
|
Tangoo Township, Bago Region |
An. maculatus |
30 |
“ “ “ |
Susceptible |
An. vagus |
30 |
|
|
|
An. philippinensis |
30 |
|
|
|
An. jamesii |
30 |
|
|
|
An.culicifacies |
30 |
|
|
|
An. barbirostris |
30 |
|
|
|
Shwe Kyin Township Bago Region |
An minimus |
21 |
“ “ “ |
Susceptible |
An. jamesii |
30 |
|
|
|
An. philippinensis |
30 |
|
|
|
An. kawari |
30 |
|
|
|
An. vagus |
30 |
|
|
|
An maculatus |
30 |
|
|
|
An. barbirostris |
30 |
|
|
|
Beelin Township Mon state |
An. jamesii |
30 |
“ “ “ |
Susceptible |
An. maculatus |
26 |
|
|
|
An. annularis |
24 |
|
|
|
An. barbirostris |
30 |
|
|
|
An. vagus |
30 |
|
|
|
Pyin Oo lwin |
An. minimus |
30 |
“ “ “ |
Susceptible |
An. aconitus |
30 |
|
|
|
An. vagus |
30 |
|
|
|
An. maculatus |
30 |
|
|
|
An. culicifacies |
30 |
|
|
|
Taungoo |
An. minimus |
30 |
“ “ “ |
Susceptible |
An.philippinensis |
30 |
|
|
|
An. jamesii |
30 |
|
|
|
An. vagus |
30 |
|
|
|
An.barbirostris |
30 |
|
|
|
An. maculatus |
30 |
|
|
|
An. culicifacies |
30 |
|
|
|
Than Byu Zayat |
An. tessellatus |
30 |
“ “ “ |
Susceptible |
An. vagus |
30 |
|
|
|
An. supitus |
30 |
|
|
|
An. barbirostris |
30 |
|
|
Incrimination of Suspected Anopheline Vectors Using Sporozoite ELISA Methods
Anopheles mosquitoes including malaria vectors and suspected vectors of malaria were collected from Inn Daw Township Sagaing Region, Shwe Kyin and Tangoo Townships Bago Region, Than Byu Zayat and Beelin Townships Mon State, Kamamaung and Hpa-an Townships Kayin State and Pyin Oo Lwin Township Mandalay Region. Morphological identification was done according to standard Anopheles mosquito keys. A total of 3150 primary and secondary vector Anopheles mosquitoes comprising An. dirus 58, An. minimus 311, An. annularis 174, An. maculatus 384, An. philippinensis 620, An. karwari 138, kochi 61, An. tessellatus 154, An. aconitus 95, An. jamesii 145, An. vagus 503, An. culicifacies 218, An. subpictus 113, An. dirus 3, An. barbirostris 153 and An. hyricanus 78 were collected and tested with sporozoite ELISA test kit in the laboratory. An. dirus was found 1(1.72%) Pf sporozoite positive and An. minimus was found 2(0.64%) Pv210 sporozoite positive. Other remaining secondary vectors were found no positive reaction was detected from mosquito samples from all study areas.
Sibling Species Identification
Sibling species identification of collected An. dirus, An. minimus and An. culicifacies were done Ovarian nurse cell polytene chromosome method and found that collected An. dirus from Than Byu Zayat Township, Mon State and Kamamaung Township Kayin State were found sibling species D and An. minimus from In Daw, Tangoo, Shwe Kyin, Beelin, Than Byu Zayat, Kamamaung, Hpa-an and Pyin Oo Lwin Townships were found sibling species A. Anopheles culicifacies from Tangoo and Pyin Oo Lwin were observed as sibling species B.
Bio Efficacy of Long Lasting Insecticidal Mosquito Nets (Llins) On Malaria Vector Anopheles Mosquitoes in Malaria Endemic Areas
Malaria remains a major health problem in Myanmar. An. dirus and An. minimus are the main vectors of malaria in Myanmar. The present study aimed to determine the bio-efficacy of long lasting insecticidal mosquito nets (LLINs) on malaria vector Anopheles mosquitoes in malaria endemic areas. Therefore, Than Byu Zayat and Beelin Townships from Mon State, Shwe Kyin and Tangoo Townships in Bago Region, Kamamaung and Hpa-an Townships in Kayin State, Pyin Oo Lwin Township in Mandalay Region and In Daw Township in Sagaing Region were selected to test the bio-efficacy of used LLINs nets. Anopheles mosquitoes were collected from each Township by animal bait K-net method for 7 days. Twenty five households were randomly selected from each Township and one each used LLINs net was collected from these houses of the Townships.
Table 2: Bio-efficacy of long lasting insecticidal mosquito nets (LLINs) on malaria vector Anopheles mosquitoes in different areas.
Sr. No. |
Different brands of LLINS nets |
Than Byu Zayat |
Beelin |
Tangoo |
Pyin Oo Lwin |
Kama maung |
Hpa-an |
Shwe Kyin |
Inn Daw |
Total |
% Sensitivity |
1 |
PermaNet 2.0 |
8 |
6 |
6 |
6 |
8 |
6 |
6 |
8 |
54 |
100 |
2 |
DAWA TANA |
6(1) |
4 |
3 |
3 |
3 |
3 |
3 |
3 |
28(1) |
96.43 |
3 |
Yorkool |
3 |
3 |
4 |
2 |
2 |
3 |
2 |
4 |
23 |
100 |
4 |
BASF(Thailand) |
4 |
6 |
4 |
2 |
5 |
3 |
4 |
4 |
32 |
100 |
5 |
Net protect(India) |
3 |
3 |
3 |
2 |
2 |
3 |
2 |
3 |
21 |
100 |
6 |
Yahei (China) |
1 |
3 |
5 |
10(1) |
5 |
7 |
8 |
3 |
42(1) |
97.62 |
7 |
Total LLINS nets |
25(1) |
25 |
25 |
25(1) |
25 |
25 |
25 |
25 |
200(2) |
99 |
|
% sensitivity |
96 |
100 |
100 |
96 |
100 |
100 |
100 |
100 |
99 |
(Table 2) Shows that a total of 200 LLIN nets (25 each) were collected from Than Byu Zayat, Beelin, Shwe Kyin, Tangoo, Kamamaung, Hpa-an, Pyin Oo Lwin and Inn Daw Townships for testing bio-efficacy of LLIN nets. Of this 54 PermaNet 2.0 (Vestergard) nets, 28 DAWA TANA (Pakisthan) nets and Yorkool (China) nets, 23 BASF(Thailand), 32 Net Protect (India)(3DF) net, 42 Yahei (China) nets were collected. All collected nets were washed 0-5 times with soap powder. Bio-efficacy of LLIN nets were found to be PermaNet 2.0 100%, DAWA TANA nets 96.43%, Yorkool nets 100%, BASF(Thailand) 100%, Net protect(India) 100% and Yahei (China) 100% mortality of Anopheles mosquitoes. All tested LLIN nets were highly sensitive against Anopheles mosquitoes. One DAWA TANA net and one Yahei nets were gave 30 to 50% mortality. (WHO Cut off value is 80% mortality). Therefore, these mosquito nets were re-impregnated with Deltamethrin insecticide at the rate of 86mg/meter square in the survey.
Figure 2: Different biting behavior of main vectors An. dirus and An. Minimus.
(Figure 2) showed that different biting behavior of main malaria vectors An. dirus and An. minimus. The pick biting behaviors of main collected vectors from different areas were found at 10:00 pm for An. dirus and at 11:00 pm for An. minimus.
Table 3: Detection of Anopheles sp. in different breeding places in villages in different areas.
Larval Survey In Different Areas |
|||||||||||
Larva inspected places |
Species of mosquito larvae |
TBZ |
BL |
TG |
SK |
KMM |
PA |
ID |
POL |
Total larvae |
% |
Sand pools
|
An. minimus, An. culicifacies
|
8 |
5 |
6 |
9 |
25 |
8 |
12 |
37 |
110 |
145 (12.33%) |
4 |
0 |
0 |
0 |
12 |
0 |
0 |
19 |
35 |
|||
Water pool
|
An. annularis, An.jamesii An.tesselatus An. maculatus |
3 |
0 |
0 |
0 |
23 |
6 |
6 |
8 |
46 |
126 |
0 |
3 |
6 |
4 |
2 |
6 |
3 |
0 |
24 |
-10.50% |
||
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||
|
|
15 |
5 |
9 |
7 |
0 |
0 |
11 |
9 |
56 |
|
Slowly running water
|
An. minimus, An. maculatus An. culicifacies An.barbirostris An. aconitus
|
8 |
6 |
11 |
11 |
9 |
5 |
16 |
117 |
183 |
354 |
6 |
8 |
9 |
6 |
0 |
9 |
21 |
23 |
82 |
-29.50% |
||
0 |
0 |
6 |
0 |
0 |
0 |
0 |
3 |
9 |
|||
14 |
4 |
4 |
9 |
5 |
0 |
13 |
2 |
51 |
|||
0 |
8 |
0 |
0 |
0 |
0 |
0 |
21 |
29 |
|||
Foot prints |
An. vagus |
15 |
5 |
6 |
6 |
13 |
2 |
19 |
8 |
74 |
74 (6.17%) |
Domestic Wells |
An. dirus |
154 |
0 |
0 |
0 |
35 |
0 |
0 |
0 |
189 |
189 (15.75%) |
Rice fields
|
An.minimus An. maculatus An.vagus An.philippinensis An.aconitus |
12 |
5 |
8 |
7 |
10 |
6 |
14 |
47 |
109 |
312 |
8 |
4 |
7 |
6 |
0 |
3 |
11 |
16 |
55 |
-26% |
||
13 |
8 |
3 |
3 |
3 |
2 |
9 |
1 |
42 |
|||
25 |
6 |
6 |
6 |
8 |
3 |
23 |
0 |
77 |
|||
|
6 |
0 |
0 |
0 |
0 |
0 |
23 |
29 |
|
||
Total
|
285 -23.75% |
73 -6.08% |
81 -6.75% |
74 -6.17% |
145 -12.08% |
50 -4.17% |
158 -13.17% |
334 -27.83% |
1200 -100% |
1200 -100% |
(Table 3) shows that highest density of larvae were found in slowly running water (29.5%) in which An. minimus183, An. maculatus 82, An. culicifacies 9, An. barbirostris 51, and An. aconitus 29 larvae were collected and they were found co-breeder in slowly running water, followed by Rice fields (26.00%) in which An. minimus (109), An. maculatus(55), An. vagus(42), An. philippinensis(77) and An. aconitus (29) larvae were collected and they were co-breeders in rice field water. The lowest density of larvae was collected in foot print of the cows, buffaloes and human (6.17%), only An. vagus larvae were observed. An. dirus was found in domestic water wells in high density (189, 15.75%) and An. minimus was found high density in slowly running water in Pyin Oo Lwin Township.
When compared with each township, highest the larval density was found 334 (27.83%) in Pyin Oo Lwin Township followed by 285(23.75%) in Than Byu Zayat Township and lowest larval density was observed in 50(4.17%).
Discussion
In these study areas 80% of the total population are farmer, woodcutter, charcoal baker, hunter, and fisher man and about 60% of the geographical areas is forested foot hill areas and malaria is endemic. Study carried out in different areas reported An. dirus culicifacies, An. vagus, An maculatus, An. annularis and An. philippinensis are the secondary or suspected vectors in Myanmar [1].
Present study found that high prevalence and dominantly present of main vector An. minimus in all selected areas such as Inn Daw, Tangoo, Shwe Kyin, Beelin,Than Byu Zayat,Hpa-an, Kamamaung and Pyin Oo Lwin Township. Main vectors An. dirus was collected from Than Byu Zayat and Kamamung Townships Mon and Kayin States. Other researchers reported that high density of An.dirus were collected from Oktwin Township Bago Region and Tanin thayi Region and low density of An. dirus was observed in Kamamaung Township Kauin State [18,19,20]. Anopheles minimus was collected in all study areas although the highest density of An. minimus (23%) was found in Pyin Oo Lwin Township followed by Tangoo (13%) and lowest density was found (4%) in Inn Daw Township Sagaing Region. Same result has been found in Tangoo, Oktwin Townships in Bago Region, Kamamaung Township in Kayin and Pyin Oo lwin Township in Mandalay Region [21,18,20]. Only 1 to 3 % of An. dirus was collected from Kamamaung and Than byu Zayat Townships. Previous study in same area mention that high number of An dirus and An. minimus adult were collected [2] . Other researcher observed that high density of An dirus and An. minimus were collected in hilly, plain and coastal areas in Myanmar [1,18,19,20, 22]. Secondary vectors Such as An. maculatus, An. aconitus, An.annularis,and other An, philippinensis, An. vagus and An. jamesii were collected in high percentage in all areas. Khin Maung Kyi revealed that secondary vectors are abundemtly present in all parts of Myanmar such as Bago, Mon , Kayin, Kayah, Taninthayi and Sagain Region [1]. An.annularis was high in Kamamaung, Hpa-an, Pyin oo Lwin, and Beelin Townships and low density was observed in Inn Daw Township. High density of An. annularis were found in Rakhine State and they are local vector of malaria in this area[1]. An. philippinensis was found high density in Inn Daw(40%) followed by Kamamaung(18%) and lowest was observed (13%) in Hpa-an Township. An. philippinensis is a secondary vector of malaria in Myanmar although this species are main vector of malaria in India and Philippine [23]. High number of An. culicifacies was collected from animal bate than indoor and outdoor light trap collection. Anopheles culicifacies is a secondary vector of malaria and sibling species B, this species was abundently present in Paukkaung and Madaya Townships in Myanmar [24].This is due to present of paddy fields, slow running streams, sand pools water pools in stream bank and stream channels around the villages are the referred breeding sites of above Anopheles mosquitoes [13,18,25] .In the present study An. dirus was found 1(1.72%) Pf sporozoite positive and An. minimus was found 2(0.64%) Pv210 sporozoite positive. These sporozoite positive was observed in Kamamaung Township Kayin State. Previous study in Kayin state found observed that 1.07% of An. minimus was Pf sporozoite positive in Katine Htit village and 3.7% of An. dirus was Pv210 sporozoite positive in Kaine Taw village[19,22]). An. minimus is a main vector of malaria in forest foothill and plain areas and An. dirus is a main vector of malaria in forested areas of Myanmar [1] and they are main vector of malaria in Assam India [23]. A study of Yesetkan village Taikky Township also found sporozoite positivity in salivary gland of An. minimus and An. maculatus by ELISA method [26]. Plasmodium vivax is a malaria parasite and it was gradually rising in some parts of Myanmar [27] Anopheles nuneztovari is an important vector of Vivax malaria in other parts of South America [28] and was incriminated as a vector of P.v210 in the Brazilian amazon [29]. Other remaining secondary vectors were found no positive reaction was detected from mosquito samples from all study areas. Tun Lin and his associates revealed that An. dirus and An. vagus were found sporozoite positive by ELISA method in Oktwin Township Bago Region [18] . Another studied in Rakhine and Taninthayi Region observed that An. culicifacies in Rakhine State and An. kochi, An. minimus, An. dirus and An. maculatus were found to be circum sporozoite positive in Taninthayi Region [30]. Monitoring of insecticide susceptibility status in some Anopheles were conducted in Inn Daw Township in Sagaing Region, Beelin and Then Byu Zayat Townships in Mon State, Tangoo and Shwe Kyin Townships in Bago Region, Hpa-an and Kamamaung Township in Kayin State and Pyin Oo Lwin Township in Mandalay Region using WHO test kits. All collected Anopheles were found no insecticide resistance detected, all were susceptible to Deltamethrin 0.05%, Permethrin 0.75% , Cyfuthrin 0.15% and DDT4%. Anopheles mosquitoes collected from Bago, Mon, Taninthayi, Kayin, Kayah, Shan, Magwe and Mandalay were susceptible to WHO recommended impregnated insecticides [31,22] . A total of 200 LLIN nets (25 each) were collected from Than Byu Zayat, Beelin, Shwe Kyin, Tangoo, Kamamaung, Hpa-an, Pyin Oo Lwin and Inn Daw Townships for testing bio-efficacy of LLIN nets. Of this 54 PermaNet 2.0 (Vestergard) nets, 28 DAWA TANA (Pakisthan) nets and 23 Yorkool (China) nets, 32 BASF(Thailand), 21 Net Protect (India)(3DF) net, 42 Yahei (China) nets were collected. All collected nets were washed 0-5 times with soap powder. Bio-efficacy of LLIN nets were found to be PermaNet 2.0 100%, DAWA TANA nets 96.43%, Yorkool nets 100%, BASF(Thailand) 100%, Net protect(India) 100% and Yahei (China) 97.62% mortality of Anopheles mosquitoes. All tested LLIN nets were highly sensitive against Anopheles mosquitoes. Therefore this mosquito net was re-impregnated with deltamethrin insecticide at the rate of 86mg/meter square in the survey. Results of earlier bio-efficacy studies of LLINs and ITNs bed nets shows that Deltamethrin impregnated nets cause 100% mortality on unwashed nets as examined by WHO tube and cone test [32,33] Maung Maung Mya and his party mentioned that PermaNet 2.0, Yarkool, BASF, Net protect, Yahei LLINs Nets and DAWA TANA nets were found 100% and 91.67% efficacy [33]. Other study found that washing does not alter the insecticidal effectiveness of PermaNet2.0, Power net and K-O-Tab123® net and insecticidal potency recovers within 24 hours and also reported that insecticidal effect in ITNs against malaria vectors remains there even after 20 months hand washing including transmission period [34,35,36]. Jaramillo and his party revealed that the regeneration study of PermaNet® 2.0 showed that efficacy was fully recovered by 24 h after one and three washes and wash resistance persisted for 15 washes. Regeneration of Olyset® nets was not observed for nets washed three times, even with the different temperature exposures for up to seven days [37]. Delenasaw and his associates found a wider range of maximum mortality i.e. 13.9% - 81.1%, in their study on 6 WHO recommended LLINs in South-Western Ethiopia [38]. LLINs are still the main malaria vector control priority based on WHO recommendation [39]. Head and thorax samples were found to be no naturally infected with Plasmodium species. An. culicifacies has been reported to be more exophilic than endophilic with low man hour biting densities in outdoor and indoor collection and found highly zoophilic in nature [40] Very low number of An culicifacies B was collected resting indoors in human dwellings in morning collection. Similar result has been found in Chhattisgarh and Orissa in India, An. culicifacies and An. fluviatilis were found resting in human dwelling as the cattle sheds were open type without walls [41,42]. Vector incrimination study revealed that only one An. minimus pool sera was positive for pv247 by ELISA method. None of the pools specimens of An. culicifacies B species was found with circumsporozoite antigen of P. falciparum and P. vivax 210 and P. vivax 247. Although vector incrimination study of An. culicifacies B in India and Pakistan revealed that An. culicifacies B is poor vector of malaria in India [43] and High number of An culicifacies B was found sporozoite positive by vector incrimination study in laboratory [44]but the species B is a main vector of malaria in Srilanka [45]. Other researchers from different countries revealed that two forms of Anopheles minimus, designated A and B, were described in China [46,47] based on morphological features in larvae, pupae and adults [48] and An. minimus A and B was presented in Thailand [49], it was confirmed in Vietnam [50](Van Bortel et al., 1999). Recently, Chen et al.,[51] showed that forms A and B in China are morphological variants of Anopheles minimus A. In Japan, Somboon et al. [52] provided morphological, cytogenetic, and molecular revealed another sibling species, designated species E, on the Ishigaki Island of the Ryukyu Archipelago. Van Bortel et al., [50] mentioned that Anopheles minimus A and C species in Northern Vietnam and found the absence of the HP (Humeral pale spot) was common (99%) in Anopheles minimus A and common (92%) in Anopheles minimus C. Another study in different areas revealed that An. dirus D in Mon and Kayin States and An. dirus A in Sedawgyi Township Mandalay Region were observed and An.minimus A was observed in PyinOoLwin Township Mandalay Region and Kamamaung Township Kayin State [22,53]. Vector incrimination study of other pools samples of suspected vector of Anopheles mosquitoes as An. maculatus, An. vagus, An. annularis and An philippinensis were found negative for circumsporozoite antigen of P. falciparum and P. vivax210 and P. vivax247 by ELISA method. An. maculatus was recently reported as important vector of malaria from research conducted Bokpin Township, Thaninthayi Region near the Thai border [30]. Tun Lin et al., [18] revealed that An. vagus and An. dirus collected from Thabwewa village Oktwin Township were found circumsporozoite antigen positive by vector incrimination study using ELISA technique. Maung Maung Mya and his party revealed that suspected vectors are predominantly present in both Bawbin dam, Ghopinkauk Township Bago Region and Maybinther village Myothit Township Magwe Region, and all collected An. culicifacies from both areas was found sibling species B by polythene chromosome study and also vector incriminated study showed they are non-vectors of malaria in these areas because all tested An. culicifacies pools samples have not positive for circumsporozoite antigen [54]. All four malaria species, P. vivax was circulating in the An. minimus. It was the most important malaria vector in Bowbin dam areas of Bago Yoma foothill area in Ghopinkauk Township Bago Region due to lack of An. dirus in this area in the study period. The collected mosquito samples from Maybinther were found negative for sporozoite antigen [54]. In the present study pick biting time of An. dirus and An. minimus from different areas were found An. dirus was at 10:00 pm and the An. minimus was at 11:00 pm. Although a bionomics study in Ye Township revealed that pick activity was found 22:00 to 23:00 hour in both Koe Main and Man Aung villages. Only four An. minimus from Koe Main and one An. minimus from Man Aung were collected in indoor between 21:00 to 24:00 hour [55]. Although Thailand researchers revealed that An. minimus showed biting activity at 19:00-21:00 hour in indoors and at 19:00-20:00 hour and 23:00-00:00 hour in outdoors [56]. In a previous study in western Thailand, the main biting activity of An. minimus was indoors between 01:00 and 04:00 with a peak at 02:00, whereas outdoor feeding started around 22:00, reaching a peak near midnight [57]. Other researcher revealed that An. dirus and An. minimus were highly bite between 10 to 11 pm and pick bite was found indoor at 11 pm [18,19,54]. The main vector of An.minimus larvae were collected from slowly running water and rice fiend water and secondary vector as An. maculatus, An. culicifacies, An.aconitus were found co breeders in these water sources. Main vector An.dirus larvae were collected from water wells in Thanbyu Zayet, Beelin in Mon state and Kamamaung in Kayin state. The lowest density of An.vagus larvae were collected in foot print of the cows, buffaloes and human. An.minimus was found high density in slowly running water in Pyin Oo Lwin Township Mandalay Region, Kamamaung Township Kayin State and Taikkyi Township in Yangon Region [22,2,58]. Sporadic studies on different species of mosquitoes suggest ecological and biological differences in types of larval habitat, seasonality and behaviour according to species [59]. Breeding habitats of different mosquito species in Koe Mine, Man Aung and 14 Mine (Ma Hlew Taung) villages in Ye Township Mon State were found An. minimus, Culex and Aedes larvae bred in domestic water wells [55]. Even so, Anopheles species exploit a variety of breeding habitats that vary considerably in size, altitude, vegetation cover and topography [60] . Although other researchers revealed that An. dirus larvae were highly breeding in water wells in Mon State and Taninthayi Region [18,19]. But they are deep forested breeding in rock pools in Thabewa village Oktwin Township Bago Region [18]. Maung Maung Mya and his associate reported that An. dirus larvae were found in water wells and An.minimus larvae were found in sand pools and slowly running water of Yunsalin in Kamamaung Township in Kayin State and domestic water wells in Ye Township in Mon State in rainy season [19,61]. In a coastal village, Chantaburi Province of Thailand near the Cambodian border, the breeding of An.dirus is in small water-collections (gam pits) [62].
Conclusion
In conclusion the main vector of An. minimus was found almost all of the study areas in high density and An. dirus was found in Kayin and Mon States in moderate density. Different sibling species such as An. minimus A, An. dirus D and An. culicifacies B were collected in study areas these were primary and secondary vectors of malaria. High density of An. minimus larvae and An. dirus larvae as well as secondary vector such as An. maculatus, An. culicifacies and An. vagus were collected in studied areas, it is a high risk for malaria transmission although All collected Anopheles mosquitoes were found susceptible to WHO recommended insecticides as Deltamethrin 0.15%, Permethrin 0.75%, Cyfuthrin 0.15% and 4% DDT and the bio-efficacy of different brands of LLINs nets were found higher efficacy to kill and prevent the mosquitoes bite and reduce the mosquito-density as well as reduce the men vector contact. They could either complement or replace some of the preventive and protective measures against the mosquito compare to other mosquito control techniques. Based on the present study, it is apparent that LLIN-based intervention technology is effective and appropriate for the control of malaria-transmitting Anopheles mosquitoes that they are abundantly present in malaria endemic areas in Myanmar and this method is a greater community acceptance, compliance and retention. Although, LLINs are needed for prolonged net serviceable lives and needed to develop effective LLINs nets that are more robust and potent against multiple insecticide-resistant insects. Study strongly recommended that LLINs nets are most effective mosquito control tools for controlling malaria transmission in community.
Acknowledgements
I am thankful to WHO because the study was supported by WHO/APW grants from World Health Organization SEARO via to Regional Office of WHO Myanmar. I am thankful to my Director General and Deputy Director Generals who allowed me to do the research. I am also highly thanks to authority concerns who were permitted and helpful to do the research works in their villages. And also I am thankful to the staffs of Medical Entomology Research Division, Department of Medical Research and Vector Born Diseases Control Program who were helpful to do the research works till completion.
Conflict Of Interest
No conflict of interest.
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