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

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.

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)

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.

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.

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.

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

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.

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.