Myanmar Health Sciences Research Journal
Original Articles :
Myanamr Health Research Registration 2020; 32(2): 120-124.
DOI: DOI: https://doi.org/10.34299/mhsrj.00991

Detection of Naturally Infected Wolbachia Bacteria in Dengue Vector Aedes Mosquitoes in Upper Myanmar

Mya Nilar Chaw Su, Yi Yi Mya, Myint Myint Aye, Nwe Nwe Kyaw & Aung Kyaw Kyaw

Myanmar Health Sciences Research Journal, 2020; 32(2):120-124

ABSTRACT

The endosymbiotic bacteria in the genus Wolbachia have been focused as a potential candidate to control medically important vector mosquitoes. Before the application of Wolbachia-based vector control strategies, it is crucial to understand Wolbachia-mosquito interaction. This study aimed to determine the infection status of Wolbachia in Aedes aegypti and Aedes albopictus from different geographical regions and it was conducted during 2018-2019. Mosquitoes were collected by using standard Ovi-traps collection method in four different townships (Mandalay, Pyin Oo Lwin, Monywa and Lashio), Upper Myanmar. Collected mosquitoes were morphologically identified to select Ae. aegypti and Ae. albopictus. A total of 320 mosquitoes were screened for the presence of Wolbachia by using polymerase chain reaction (PCR) method. Among them, male and female were distributed equally in both species. Result revealed that 67.5% (108/160) of Ae. albopictus were observed as positive Wolbachia infection.  However, no infection was detected in dengue primary vector Aedes aegypti. Wolbachia infection of super groups A, B and super infection of A+B was found in Ae. albopictus. Wolbachia Group B infection is higher than Group A infection in the study populations. Wolbachia infection was found in every parts of study areas. However, further study is still required to explore the role of bacterial-infected mosquitoes for vector-borne disease control activities.


RESULT
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A total of 320 mosquitoes samples collected from eight collection sites in four townships were screened for the Wolbachia infection using wsp gene primer.No Wolbachia infection was detected in Ae. aegypti.Result showed 67.5% (n=160) of Ae. albopictus had positive infection of Wolbachia bacteria,whereas 38.1% of female and 29.4% of male had positive infection (Table 1).

 

Table 1.Infection status of Wolbachia from field collected Ae. albopictus mosquitoes

Study site

Sex

Tested mosquito(n)

No. of Wolbachia infected
mosquito (%)

Mandalay  Region

 

 

Mandalay

Male

20

17(85.0)

Female

20

12(60.0)

PyinOoLwin

 

Male

20

15(75.0)

Female

20

18(90.0)

Sagaing Region

 

 

Monywa

Male

20

10(50.0)

Female

20

15(75.0)

NorthernShan State

 

 

Lashio

Male

20

5(25.0)

 

Female

20

16(80.0)

Grand total

160

108(67.5)

Super-infection of both group A+B was 39.3% and single infection of group A and B was 11.2% and 16.8%, respectively(Table 2).Super-infection of group A+B and


Table 2.Different types of Wolbachia infection among field collected Aedesal bopictus mosquitoes

Study

site

Sex

(n)

wAlbA

wAlbB

wAlbA+B

positive

(%)

positive (%)

positive

(%)

Mandalay

Male

20

8(40)

5(25)

4(20)

Female

20

-

-

12(60)

PyinOo

Lwin

Male

20

4(20)

-

11(55)

Female

20

2(10)

2(10)

14(70)

Monywa

Male

20

4(20)

4(20)

2(10)

Female

20

-

5(25)

10(50)

Lashio

Male

20

-

3(15)

2(10)

Female

20

-

8(40)

8(40)

Grand total

160

18(11.2)

27(16.8)

63(39.3)

 

only group B single infection was observed in Northern Shan State. Mandalay Region showed the highest infection rate and Northern Shan State showed the lowest infection rate in the study population.

 



INTRODUCTION
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Arboviruses transmitted by mosquitoes are a major cause of human diseases worldwide. There are many different arboviruses, in which dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus and Zika virus are medically important viruses.1 In particular, dengue is currently the most important life threatening arthropod-borne viral disease of humans and primarily transmitted by female Aedes aegypti and Aedes albopictus. Although dengue vaccine is available in some countries, there is a limitation in effectiveness on all serotypes of dengue virus.2Thus, dengue disease prevention and control is still relying on vector control measures. Four different groups of insecticides have been applied; organochlorine, organophosphates,carbamate and pyrethoids for vector control globally.3 Wide and indiscriminate use of insecticides has been created insecticide resistance problem worldwide. DDT and pyrethoids(deltamethrin and permethrin) resistance in some dengue and malaria vectors were reported in Myanmar.4,5

The alternatives of new mosquito control strategies are either suppression or replacement of mosquitoes that are unable to transmit viral diseases. Among alternative strategies,Wolbachia approach is very promising method, safe to human, cost effective and ecofriendly.6 Wolbachia are maternally inherited endosymbiotic bacteria recognized to infect a broad range (25-75%) of arthropod species and some filarial nematodes.7 Previous study in India showed 20% of five species of Aedes genus, 50% of eight speciesof Culex, 100% of two species of Armigeresand100% of one species of Toxorhynchites were infected by Wolbachia, whereas genera Anopheles and Lutiza were not positive for Wolbachia infection.8 In Malaysia, the prevalence of Wolbachia infection status of field collected Ae. albopictus was estimated to be 98.6%.9

Wolbachia strain can induce various phenotypic effects in insects including parthenogenesis, feminization, male killing and cytoplasmic incompatibility. Critically, the bacterium also blocks the transmission of many important human pathogens in mosquitoes, including plasmodium, dengue and chikungunya, giving it great utility as a control agent.10

The diversity of Wolbachia infection in mosquito genera has been well investigated to certain parts of south-east Asia, Europe and Africa.8 However, the presence of Wolbachia in natural populations of mosquitoes has never been studied in Myanmar. Thus, here the prevalence of Wolbachia infections among natural populations of dengue vectors mosquito in Myanmar was discussed and presented as
a first time in Myanmar.


SUPPLEMENTARY MATERIAL
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Mosquito collection, rearing and identification

Aedes mosquito larvae were collected by using ovi-traps in four different townships under three States and Regions, namely Mandalay and Pyin Oo Lwin (Mandalay Region), Monywa (Sagaing Region) and Lashio (Northern Shan State). For collection of natural population of Aedes mosquitoes, ovi-trap was used in the present study. As an ovi-trap, 200 ml water was filled in black colored (300 ml capacity) plastic and 12 cm length x 2 cm width bamboo stick was half submerged in the water to act as ovi-position site for mosquito. In each collection site, 30 ovi-traps were used and ovi-traps were installed at least 100 meters apart from each other to minimize the probability of progeny from same mother. One week later, ovi-traps were brought back to the insectary and Aedes larvae were collected from each ovi-trap. Collected mosquito larvae were reared in the insectary with control temperature and humidity (25±2ºC and 75±5%, respectively). Fish powder was provided to immature stages and 10% sugar solution was provided to adult mosquitoes. Newly emerged adult mosquitoes were identified morpho-logically.11 After identification, one to three days old newly emerged adult mosquitoes were individually stored at -20ºC for further study.

DNA extraction

From each study site 20 samples were randomly selected and male and female were equally involved for DNA extraction. The mosquito was individually homogenized and Chelex DNA extraction was performed.12 Extracted DNA samples were stored at -20ºC for the next step polymerase chain reaction (PCR). Before PCR, DNA purity was checked by Nano-Drop (DNA spectrophotometer, UK).Screening for Wolbachia by polymerase chain reaction Multiplex PCR was carried out using the temperature profile of 95°C for 5 minutes; 95°C for 30 seconds, 55°C for 20 seconds for 30 cycles and 72°C for 20 seconds using wsp primers for A group; ( wAlbA Forward-5̍-TGA AAT TTT AGC TCT TTT C3̍, wAlbA Reverse-5̍-AAA AAT TAA ACG CTA CTC CA 3̍ ) and for B group (wAlbB Forword-5̍-TGG TCC AAT AAG TGA TGA 3̍, wAlbB Reverse-5̍-ACC AGC TTT TGC TTG ATA3̍).8 In the negative sample 28s Forward 5̍- TAC CGT GAG GGA AAG TTG AAA 3̍ and Reverse 5̍ AGA CTC CTT GGT CCG TGT TT 3̍ primers were used for insect ribosomal locus.13 Analysis of the PCR products was conducted by gel electro-phoresis. Seven microliters of the PCR product was loaded onto 1.5% agarose gel and the amplicons were documented by gel documentation unit. Yield fragments of Wolbachia Group A and B primers were about 550 and 450 bp, respectively.


DISCUSSION
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In the present study, no infection of Wolbachia was detected in Aedes aegtpti mosquitoes. Similarly, a previous study published in 2018 reported that there was no evidence of Wolbachia infection in natural population of dengue vector Aedes aegypti mosquitoes collected from 27 countries.14In contrast,a study from Phillipine reported that 11.9% of Wolbachia was detected in a natural population of Ae. aegypti.15

In Ae.albopictus, not only Wolbachia group A and B were found separately but also super infection of A+B was found in natural population. Interestingly, proportion of Wolbachia super-infection was higher than single infection. It is possible that infection with 2 different strains of Wolbachia may contribute to the high fidelity of maternal transmission of Wolbachia in natural populations of this mosquito species.16 Wolbachia infection was higher in female Ae. albopictus than in male. Frequency of Wolbachia infection pattern was not homogenous among study population. Single infection was found in some study population and it might depend on the environment and food sources. Similar patterns had been reported on Wolbachia infection in different host from other countries.17 Kittayapong,et al. reported that frequency of Wolbachia infection was 100% in natural populations of the mosquito vector Ae.albopictus.18  Previous study in Malaysia showed 91.6% of super-infection Wolbachia A+B group and only low level of single infection 3.15% were found in Ae. albopictus.19In the present study, Wolbachia infection rates were lower than the other published results. Wang et al., mentioned that the number of Wolbachia reads was the lowest in young adult and increased substantially in older female adults.20Thus, using of young adult mosquitoes might be the reason for detection of low Wolbachia infection rate in the present study.

Interestingly, it has been found that the presence of some Wolbachia strains in mosquitoes can regulate the expression of genes involved in the immune responses, resulting in inhibition of the replication, multiplication, or resistance to the proliferation of viruses, parasites, and microfilariae.21In this sense, Wolbachia can also be visualized as a microorganism for biological control that is based on the substitution of the microbiome of the vector by microorganisms that affect vector’s pathogen load.22 Present study will provide basic descriptive information to devise experimental strategies by exploiting a Wolbachia-cytoplasmic incompatibility-based mechanism to control vectors. Myanmar is dengue endemic country and currently ASEAN countries have been tried to collaborate sterile insect technique (SIT) and Wolbachia-based control strategies in the inter-regional countries. Therefore, present study is useful to provide dengue vector control strategies.

Conclusion

Although Wolbachia infection was not observed in Ae.aegypti mosquito, Wolbachia infection of Super groups A, B and super-infection of A+B was found in Ae. albopictus. Wolbachia Group B infection is higher than Group A infection in the study populations.Importantly Wolbachia super-infection was found in every collection sites of study areas. Despite as first time detection of Wolbachia infected mosquitoes in Myanmar, the data from present study provided the important information for future vector control activities.


 


ACKNOWLEDGMENT
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We are very grateful to Director-General, Department of Medical Research and Deputy Director-General for encouragement and supporting our study. We also would like to show our appreciation to Inha University (Republic of Korea) for their technical and material supports.

 


CONFLICT OF INTEREST
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The authors declare that they have no competing interests.

 



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