Detection of Wolbachia from field collected Aedes albopictus Skuse in Malaysia

Wolbachia species are obligate intracellular rickettsia-like bacteria belonging to the alpha subclass of Proteobacteria and the order of Rickettsiales that live inside the cells of various organs, but most frequently appear in ovaries and testes1. Wolbachia infects a wide range of arthropods including mosquitoes, ticks, flies and nematodes populations2. However, some of the major disease vectors are not naturally infected, including the primary vector of dengue, Aedes aegypti and all anopheline mosquitoes sampled to date34. In arthropods, Wolbachia causes several host reproduction alterations, including cytoplasmic incompatibility (CI). In the simplest CI scenario, the mating between males and females that carry different and incompatible strains of Wolbachia will eventually result in a significant reduction in fecundity and egg hatching rate. Similar effects were also observed when mating occurred between uninfected females with infected males56. The introduction of Wolbachia from its native host into new hosts exhibited an interference with pathogens through several mechanisms including the upregulation of several immune genes78. The ability of a Wolbachia strain (wMelPop) to interfere with pathogens development and to cause reduction in adult mosquito longevity has also been reported910. From an applied perspective, Wolbachia utilizing strategies can be used for either population suppression or sweep desirable traits into pest populations such as the inability to transmit disease-causing pathogens711, given that the Wolbachia infection must reach a stable equilibrium within the target population, at a rate that is high enough to cause a significant impact and eventually reducing disease transmission by the vector12.

In Malaysia, Ae. albopictus Skuse and Ae. (Stegomyia) aegypti (Linnaeus) are the incriminated dengue vectors. The transovarial transmission of dengue virus has been proven for both species under both laboratory and field conditions1314. Despite being the secondary vector for dengue virus, under some circumstances, the transovarial transmission of dengue virus was proven to be higher in larvae of Ae. albopictus compared to Ae. aegypti larvae15. Ae. albopictus adult was reported to be responsible for the viral transmission during dengue fever outbreaks not only in its native regions but also in introduced ranges like in Hawaii16 and Mexico17. Thus, it is crucial to know the infection frequency and the types of native Wolbachia strain infections for both female and male Ae. albopictus mosquitoes, prior to exploring the potential use of CI-based strategies for dengue transmission control.

In the present study, field surveys were conducted to detect Wolbachia in both female and male field-collected Ae. albopictus mosquitoes from various habitats.

Material & Methods

Mosquitoes collection: Ae. albopictus mosquitoes were collected from five different localities ranging from housing areas (Malacca, Selangor), seashore (Terengganu) and islands areas (Ketam Island, Carey Island) in Peninsular Malaysia from March till September 2012. Adult Ae. albopictus were collected using human landing catches and hand aspirator and individually placed in glass vials. Standard ovitraps were also set in the selected areas for five days and the larvae were identified to species level using the Aedes sp. key18. The larvae from each ovitrap were placed inside an adult cage (25×25×50cm) for emergence. The adult mosquitoes emerging from each ovitrap were sexed and pooled together (2-10 mosquitoes/pool) at the age of 7-10 days. All mosquitoes were killed by keeping them in -20°C freezer for one hour just prior to pooling. The mosquitoes were homogenized with a clean pestle and incubated in 20 μl proteinase K at 56°C in a shaking water bath for three hours. The subsequent procedures were performed according to the QlAamp® DNA Mini Kit protocol (Qiagen™, Germany).

Detection of Wolbachia: Multiplex PCR was carried out using the temperature profile of 95°C for 1 min, 55°C for 1.5 min and 72°C for 2 min for 35 cycles using wsp primers. Primers used were 328F and 691R for wAlbA and 183F and 691R for wAlbB as described by Zhou19(328F, 5’-CCA GCA GAT ACT ATT GCG-3’ 183F,5’-AAG GAA CCG AAG TTC ATG-3’ 691R, 5’-AAA AAT TAA ACG CTA CTC CA-3’). A negative and positive control for the PCR assay were included in each run. The positive control was obtained by screening the adult Ae. albopictus (resident strain) using PCR and sequencing of wsp gene to confirm that the amplified PCR product obtained was Wolbachia. The quality of DNA extraction was checked by running all the negative samples using 12sRNA primer set (12SA, 5’-AAA CTA GGA TTA GAT ACC CTA TTA T-3’ 12SB, 5’ - AAG AGC GAC GGG CGA TGT GT-3’)20. Any sample that was negative for both wsp and 12sRNA primer sets was excluded from the data set. Samples that were negative for wsp primers but positive for 12sRNA primers were scored as uninfected. All the positive PCR products were visualized under 1.5 per cent agarose gel electrophoresis.

Sequencing of Wolbachia endobacterium: The positive PCR product was purified using QIAquick® Gel Extraction Kit (Qiagen, Germany) prior to DNA sequencing. One DNA extract each from individual and pooled mosquitoes for each locality (Malacca, Carey Island, Ketam Island, Terengganu and Selangor) was outsourced for sequencing. All sequences were subjected to run in Basic Local Alignment Search Tool (BLAST®) (retrieved from: (http://blast.ncbi.nlm.nih.gov/) available in the GenBank.

Multiple sequence alignment was carried out using Clustal-W programme21. The evolutionary distances of Wolbachia isolates from Ae. albopictus based on wsp genes were constructed using Neighbour-Joining tree using Kimura-2P analysis with 1000 bootstrap replicates in MEGA 5.1 software21. A total of seven sequences of Wolbachia strains derived from GenBank were included in the analysis.

Results

Multiplex PCR of Wolbachia in adult mosquitoes: Individual field-collected adults, as well as pools of mosquitoes recovered from positive ovitraps, were screened for the presence of Wolbachia. A total of 104 individual adult Ae. albopictus showed a 100 per cent positivity rate for Wolbachia infection; both males (n=46) and females (n=58) from the population were studied. For Ae. albopictus collected from ovitraps, it was decided to screen for the presence of Wolbachia from Ae. albopictus in pool due to the high numbers of samples recovered from positive ovitraps. A total of 73 of the 76 pools (female) and 83 of the 87 pools (male) of mosquitoes were positive for Wolbachia (Table I). To ensure that the negative pools (4 pools for female and 3 pools for male) were true negatives and not due to the poor quality of the extracted DNA, these samples were further analysed using 12sRNA primers. Based on PCR results, all the wsp negative pools were positive for 12sRNA primers, indicating the good quality of the extracted DNA and were scored as negative Wolbachia infection.

Table I Infection status of Wolbachia from field collected Ae. albopictus as determined by standard multiplex PCR.

Sequencing of Wolbachia endosymbiont DNA in Ae. albopictus: For the wsp gene, all the individual females (n=58) tested were found to be superinfected with both strains A and B Wolbachia. On the other hand, the individual male mosquitoes showed a mix of either double or single infection; A and B (n=15) or B (n=31) strain only. None of the samples screened was infected nor infected with strain A only (Table I).

For the mosquitoes extracted in pool, the female pools were superinfected with both strains A and B in all instances. As none were found to be single infection with strain A or strain B only in individual females screened, it was inferred that the superinfection of strains A and B in the female pool was true. On the other hand, the pools for male mosquitoes showed a mix of double and single infection of strains A and B or strain B only, similar to observation in individual male samples (Table I). A total of 59 pools were infected with only strain B Wolbachia, while only 24 pools of the remaining positive pools were infected with both strains A and B. However, with the pool extracted mosquitoes, the presence of A and B strains might also be contributed by strain B only from individual male mosquito in the pool and thus underestimating the presence of strain B only. The comparison between the proportion of A and B and B only infection (approximately 1:2 ratio) was relatively similar for individuals and pooled males and thus, giving the rough estimation of the types of Wolbachia infection in the population studied.

DNA of Wolbachia isolates in this study was successfully amplified and sequenced from 10 specimens (each isolate representing each locality) (Table II). The new sequences of wAlbA and wAlbB from individual samples obtained were deposited in GenBank (accession number KC004024 and KC004025). The sequencing results for wsp gene yielded fragments of 341and 463bp for strains A and B, respectively. The Wolbachia strains A and B of Ae. albopictus showed a 100 per cent homology with Wolbachia sp. in Ae. albopictus from different geographical regions. Phylogenetic analysis showed that Wolbachia isolate from the present study was closely related to Wolbachia isolated from Ae. albopictus collected from different locations and the sequences were grouping together according to the wsp strains A and B, respectively (Figure).

Table II Voucher no., accession no. and collection location for the Wolbachia strain A and B of Ae. Albopictus.

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Figure

Neighbour-joining phylogenetic tree of Wolbachia strain, isolated from Ae. albopictus based on partial sequence of wsp gene using Kimura-2P analysis. Figures in parentheses are GenBank accession numbers.

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Discussion

In arthropods, Wolbachia can cause numerous reproductive alterations. Among all the reproductive modifications caused by Wolbachia, CI has been utilized as one of the tools to control mosquitoes. CI is a form of sterility in which if the same and compatible Wolbachia strain is not present in the egg during embryogenesis, embryonic development will be disrupted1. Thus, the sustained repeated release of cytoplasmically incompatible Wolbachia infected mosquitoes will result in the increasing rate of incompatible mating and hence, lead to suppression of the vector population22.

In our study, a 100 per cent positivity for Wolbachia was observed in field-collected individual male and female Ae. albopictus. Both theoretical and empirical data have suggested that Wolbachia are expected to rapidly spread to fixation once a Wolbachia infection enters a population72324. The wsp sequencing indicated that all the female (individuals and pools) Ae. albopictus tested were superinfected with both strains A and B. This result was similar with many studies which stated that the superinfection with both strains A and B in field population of female Ae. albopictus was common and virtually fixed in a population, suggesting efficient vertical transmission of both strains A and B2526. In addition, the presence of both strains A and B in this mosquito species has been claimed to contribute to high fidelity of maternal transmission of Wolbachia27.

In most instances, the male Ae. albopictus were infected with strain B only and in some instances, they were superinfected with both strains A and B. The efficient transmission of both strains A and B observed in female Ae. albopictus advocates that the loss of wAlbA is hardly adaptive and, therefore, the same vertical transmission of both strains are expected to be seen in male mosquitoes as well. The density of type A Wolbachia has been shown to be significantly decreased with age in male Ae. albopictus population28. In some cases, the density of strain A was reduced towards a complete loss within 5-day period post-emergence. In other instances, all tested males showed no strain A infection regardless of age, suggesting that the loss of wAlbA might have taken place earlier before the emergence28. This could be the reason for the lack of strain A infection in the male population aged 7-10 days (pooled mosquitoes) as observed in this study. This observation was further supported by another study that showed a reduction in wAlbA CI in 10-days old wAlbA mono-infected laboratory reared Ae. albopictus which possibly correlated with the lack of wAlbA strain in aged Ae. Albopictus29.

Wolbachia-based vector control strategy to complement the existing method for vector control has been investigated in several laboratories113031. Though the impact of Wolbachia on mosquito vector in arbovirus transmission has been reported91131, this was not examined in our study. Our data on Wolbachia natural infection provide important initial baseline information in developing potential strategies prior to exploring the possibility of utilizing this strategy for dengue transmission control.