Introduction
Aedes aegypti and Aedes albopictus are competent vectors for dengue virus (DENV) and chikungunya virus (CHIKV). The latter also known as Asian tiger mosquito always receive(s) lesser attention as it is considered the bridge vector to Ae. aegypti-dominated urban epidemics.1 Nonetheless, the Asian tiger mosquitoes can still act as the principle vector in epidemic areas where Ae. aegypti is present.1,2 The worldwide expansion of the geographic range of Ae. albopictus makes this invasive vector of human pathogenic viruses a major concern in many locations.1 In 2005, Ae. albopictus was incriminated as a sole vector responsible for causing chikungunya outbreak of unprecedented magnitude in the Indian Ocean.3 The outbreak continued to spread to central Africa,4 India,5 and then towards Europe,6 Asia,7–10 and North America.11 In Malaysia, a nationwide outbreak occurred in 2008, starting in Johor State, which later spread to other states and federal territories affecting about 10,000 people.9,12,13 Phylogenetic analysis of the viral sequence isolates revealed a point mutation of alanine to valine at point 226 (A226V) of the E1 gene of the polyprotein, enhancing the CHIKV replication and transmission efficacy in Ae. albopictus.14,15
Wolbachia species are obligate intracellular bacteria that infect a wide range of insects as well as some species of nematodes, making it the most ubiquitous bacteria yet described.16,17 Wolbachia infection has also been detected in mosquitoes including Ae. albopictus but is not found in Ae. aegypti. Wolbachia are vertically transmitted from infected females to their progeny. Wolbachia can alter the reproduction of its host in various ways, one such way is cytoplasmic incompatibility (CI). 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 disrupted.18,19 CI phenomenon gives a reproductive advantage to the infected females, at which they can mate successfully with both infected and uninfected males and hence enhances Wolbachia invasion in a population. Wolbachia has drawn much attention as some of the Wolbachia strains (e.g., wMelPop and wMel+wAlbB) have shown to reduce mosquito life span and/or induce pathogen blocking effects on the invertebrate hosts. These effects can substantially reduce the risk of pathogen transmission.20,21
Nonetheless, the Wolbachia-mediated viral blocking effect is not ubiquitous. Unlike the case for transinfected hosts, effect of Wolbachia on virus replication in native hosts has been reported to be inconsistent. For instance, the naturally occurring Wolbachia of Aedes notoscriptus do not induce DENV interference within the native hosts22 contrary to the report on Ae. albopictus that demonstrated that native Wolbachia can limit transmission of DENV.23 Another study on Drosophila demonstrated that native Wolbachia render pathogen resistance toward the RNA viruses in their original hosts.24–26 The Wolbachia-based vector control strategies have taken the form of either population replacement or the incompatible insect technique (IIT) strategy. The population replacement strategy is highly dependent on the ability of the Wolbachia to invade and replace the target population with a population that cannot transmit virus.27,28 On the other hand, the IIT approach involves a continuous inundated release of males carrying an incompatible Wolbachia strain with that in the existing mosquito population, to suppress mosquito numbers below a threshold that enables continued virus transmission.29,30 In this study, we aim to determine the Wolbachia infection in field-collected Ae. albopictus from different geographical regions. This study is crucial to cater for the scarcity of information on Wolbachia infection status in field-collected Ae. albopictus population in Malaysia. Furthermore, we also investigated the effects of the naturally occurring Wolbachia on the replication of CHIKV in Ae. albopictus. These findings will help to facilitate the understanding of the Wolbachia–CHIKV–Ae. albopictus interaction, which will serve as a platform for the Wolbachia-based vector control approach to be conducted in Malaysia.
Methods
Mosquito collection.
Aedes albopictus was collected from eight collection sites from five states in Malaysia as shown in Figure 1. A minimum of 50 ovitraps were set in each location for 5 days and were at least 150 m apart to minimize the probability of progeny from the same mother. Aedes ovitrapping was conducted following the guidelines of Ministry of Health, Malaysia, on ovitrap deployment.31 Ovitraps were brought back to the insectarium in the Medical Entomology Unit, Institute for Medical Research (IMR), Kuala Lumpur. Eggs collected were hatched and larvae (L3) recovered were individually identified to species level according to the key by Mahadevan and others.32 The identified Ae. albopictus larvae from each ovitrap were placed into a plastic container and were supplied with liver powder. Once the larvae reached pupal stage, the plastic containers were placed inside an adult cage (25 × 25 × 50 cm). Mosquito species were again confirmed. Adult mosquitoes were supplied with 10% glucose incorporated with liquid B-complex (Atlantic Laboratories Corp. Ltd, Bangkok, Thailand) and maintained using standard condition of 28°C with 70–80% of relative humidity.
Map of Peninsular Malaysia showing Aedes albopictus collection sites. Samples were collected from eight different collection sites (indicated by red stars) from five states in Peninsular Malaysia.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
Map of Peninsular Malaysia showing Aedes albopictus collection sites. Samples were collected from eight different collection sites (indicated by red stars) from five states in Peninsular Malaysia.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
Map of Peninsular Malaysia showing Aedes albopictus collection sites. Samples were collected from eight different collection sites (indicated by red stars) from five states in Peninsular Malaysia.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
DNA extraction.
Adults (F0) aged between 7 and 10 days were subjected to DNA extraction. Briefly, 4–10 mosquitoes (males and females) recovered from each ovitrap were killed by placing them in freezer for an hour. The mosquitoes were individually homogenized in 180 μL cell lysis solution (ATL Buffer) and incubated in 20 μL proteinase K at 56°C in water bath for 3 hours. The subsequent procedures were performed according to the QlAamp® DNA Mini Kit protocol (Qiagen™, Hilden, Germany).
Detection of Wolbachia.
Multiplex polymerase chain reaction (PCR) was carried out using a temperature profile of 95°C for 15 seconds, 57°C for 30 seconds, and 72°C for 1 minute for 35 cycles using wsp primers. Primers used were 328F and 691R for wAlbA strain and 183F and 691R for wAlbB strain as described by Zhou33 (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′). The PCR mixture contained 5 μL of extracted DNA, 12.5 μL of MyTaq™ Mix (Bioline, Taunton, MA), 1 μL of each primer (10 μM), and 4.5 μL of ddH2O. Negative and positive controls 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 the extracted DNA was checked using the 12S rRNA primer sets (12SA, 5′-AAA CTA GGA TTA GAT ACC CTA TTA T-3′; 12SB, 5′-AAG AGC GAC GGG CGA TGT GT-3′) to screen samples that were negative for wsp primers using the temperature profile of 95°C for 15 seconds for denaturation, 47°C for 30 seconds for annealing and 72°C for 1 minute for extension, conducted for 35 cycles. Samples that were negative for wsp primers but positive for 12S RNA primers were scored as uninfected. All the positive PCR products were visualized under 1.5% agarose gel electrophoresis.
Sequencing of Wolbachia endobacterium.
The positive PCR product was purified using QIAquick® Gel Extraction Kit (Qiagen™) before DNA sequencing. A minimum of 10 purified DNA extracts from individuals of each locality were outsourced for sequencing. All sequences were searched against the GenBank nucleotide database using the Basic Local Alignment Search Tool (BLAST®) provided by the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Partial wsp gene sequences of Wolbachia were aligned using the Clustal-W algorithm and the evolutionary distances of Wolbachia isolates from Ae. albopictus was constructed using neighbor-joining tree, utilizing Kimura-2P analysis with 1,000 bootstrap replicates in MEGA 6.0 software.34 A Wolbachia sequence from Culex quinquefasciatus was included as an outgroup.
CHIKV production.
CHIKV (Asian strain) was provided by the Virology Unit, IMR, Kuala Lumpur. The virus was isolated during the outbreak in Bagan Panchor, Perak, in 2006. The CHIKV was maintained in BHK-21 cell lines. Stock virus prepared by freeze-thawing the infected cells once, centrifuging the suspension at 40,000 × g and storing the filtered supernatants at 80°C. The infected cells were maintained in Virology Unit, IMR. The titer of the CHIKV stock was determined using the 50% cell culture infectious dose assay.
Mosquito samples for artificial oral infection.
Mosquitoes from three localities were used for the oral infection experiment. Each locality was chosen to represent one habitat: 1) Besar Island (tourism island), 2) Tenggol Island (remote island), and 3) Bandar Rinching (urban residential area). Females from Besar Island were derived from the same collections used for Wolbachia screening. Mosquitoes from Tenggol Island and Bandar Rinching were derived from the existing colony in insectarium. All mosquitoes used for artificial oral infection were F6 generation.
Tetracycline treatment to clear Wolbachia.
Adult mosquitoes were provided with a solution of 0.75 mg/mL tetracycline dissolved in 10% sucrose. After every treatment, 10 randomly selected treated mosquitoes from each generation were tested by PCR for Wolbachia infection. Treatment was continued if any randomly tested mosquitoes were positive for Wolbachia confirmed via PCR. This treatment was performed up to four generations of mosquitoes. Colonies of Wolbachia-free Ae. albopictus were maintained for a further two generations without tetracycline before experiments commenced to allow the reestablishment of beneficial microbiota.
Experimental oral infections.
Experimental oral infection with CHIKV was conducted within an Arthropod Containment Level 2 insectarium. The artificial membrane feeding technique was performed using the Hemotek Feeding Systems (Discovery workshops, Accrington, United Kingdom) housed in an isolation glove box. Human blood used for artificial feeding was sent to Virology Unit, IMR, and confirmed to be negative by neutralization assay for CHIKV antibodies. Blood suspension containing 1:9 of CHIKV (titer 107 plaque-forming units/mL) in human blood was used for artificial feeding. Uninfected samples (control group) were obtained by feeding the mosquitoes with human blood only. A total of 250 adult mosquitoes from each group Wolbachia infected (w+) and Wolbachia-free (w−) aged 3–5 days that have been starved overnight were subjected to artificial feeding. The blood was presented to the mosquitoes by placing the cups (containing 50 mosquitoes each) below the feeder with the surface of the nylon netting of the cup in contact with the membrane of the feeder. Mosquitoes were allowed to feed for approximately 20–30 minutes. The mosquitoes in cups were cold anesthetized by placing in −20°C freezer for 30 seconds. The mosquitoes were then sorted. All unfed mosquitoes were discarded. Engorged mosquitoes were placed in cups (10 per cup) and kept in incubator at 28°C and humidity of 80% for planned time points (days 0, 1, 2, 3, 5, 7, and 10) postinoculation (PI) studies. At each time point, mosquito samples from at least a single cup were cold anesthetized by placing them in −20°C for 30 seconds, before dissection to remove midguts, salivary glands, and ovaries. Individual mosquito was put on a glass slide. The thorax and head of the mosquito were first removed, followed by dissection of the salivary glands in a drop of saline. The abdomen was then dissected to remove midguts and ovaries in a drop of saline, respectively. Glass slide was replaced for each individual mosquito, and fresh drops of saline were used for each organ examined. It was ensured that the dissecting needles were rinsed in alcohol between each dissection to prevent contamination. A total of four engorged mosquitoes fed with clean human blood at days 0 and 10 PI were kept aside to serve as negative control. For each experimental time point, the infection rate is defined as the number of midguts with detectable virus titer divided by the number of mosquitoes sampled. The dissemination rate was defined as the number of salivary glands with detectable virus titer divided by the number of midguts with detectable virus titer.
Nucleic acid extraction and quantitative PCR.
Total nucleic acid was extracted from the dissected organs (midguts and salivary glands). Extraction was performed with innuPREP DNA/RNA Mini Kit (Analytik Jena AG, Jena, Germany) that enables the isolation of both RNA and DNA. RNA was used to determine viral load by real-time reverse transcription PCR (RT-PCR), and DNA to check for the presence of Wolbachia in ovaries using conventional PCR. For w+ group, individual mosquito that was screened negative for Wolbachia (in ovaries) was excluded from the dataset. A minimum number of nine mosquitoes were used for each time point. A standard curve was generated using 10-fold serial dilutions of RNA synthetic transcript with known copy number.
Determination of limit of detection.
RNA template of known concentration was diluted using six 10-fold serial dilutions. Each concentration was run in triplicate for a total of three runs. Dilutions of CHIKV RNA ranged from 2.4 × 106 RNA copies to 2.4 × 101. The linear range was established with acceptance criteria of R2 > 0.98 with an efficiency of > 90%. The limit of detection (LOD) was defined as the lowest concentration of viral RNA that can be detected in ≥ 95% of nine replicates.
Statistical analysis.
GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, CA; www.graphpad.com) was used to construct graphs. All statistical analyses were conducted using the IBM SPSS Statistics (version 19; Armonk, NY). CHIKV infection and dissemination rates were compared with Fisher's exact test with two-tailed P values. Non-parametric statistical, Mann–Whitney U tests was used to assess the statistical differences for CHIKV titer in w+ and w− groups, and P values < 0.05 were considered statistically significant. P values were adjusted for multiple tests using the Kruskal–Wallis test with Bonferroni correction.
Results
Distribution of Wolbachia in Malaysian Ae. albopictus.
A total of 244 Ae. albopictus samples collected from eight sites in five states (Malacca, Selangor, Terengganu, Perak, and Pahang) in Malaysia were screened for Wolbachia infection using wAlbA- and wAlbB-specific wsp gene primers. Our results showed a high percentage of Wolbachia infection with 98.6% in females and 95.1% in males. For wsp gene, phylogenetic analysis revealed that Wolbachia isolates from the present study were closely related to Wolbachia isolates from different geographical regions, and the sequences were grouped into wAlbA and wAlbB clades, respectively (Figure 2). The size of the wsp fragment for wAlbA was 341 bp and wAlbB was 463 bp.
Neighbor-joining phylogenetic tree of Wolbachia strain, isolated from Aedes albopictus based on partial sequence of wsp gene using Kimura-2P analysis. GenBank sequences are shown with accession number. The new sequences of wAlbA and wAlbB from Ae. albopictus obtained in this study were deposited in GenBank with accession nos. KC004024 and KC004025 (indicated by triangle).
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
Neighbor-joining phylogenetic tree of Wolbachia strain, isolated from Aedes albopictus based on partial sequence of wsp gene using Kimura-2P analysis. GenBank sequences are shown with accession number. The new sequences of wAlbA and wAlbB from Ae. albopictus obtained in this study were deposited in GenBank with accession nos. KC004024 and KC004025 (indicated by triangle).
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
Neighbor-joining phylogenetic tree of Wolbachia strain, isolated from Aedes albopictus based on partial sequence of wsp gene using Kimura-2P analysis. GenBank sequences are shown with accession number. The new sequences of wAlbA and wAlbB from Ae. albopictus obtained in this study were deposited in GenBank with accession nos. KC004024 and KC004025 (indicated by triangle).
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
Table 1 shows the frequency of double and single infections of Wolbachia in the Ae. albopictus populations sampled from all the collection sites. For females, 97.2% (138/142) of the samples were superinfected with both wAlbA and wAlbB. Only 1.4% (2/142) of samples were singly infected with either wAlbA or wAlbB. Another 1.4% (2/142) samples were scored as uninfected. For males, 49.0% (50/102) was superinfected with wAlbA and wAlbB followed by 46.1% (47/102) infection with wAlbB only. The remaining 4.9% (5/102) samples were uninfected. None of the males were positive for wAlbA only. The uninfected samples were confirmed by running the DNA with the 12S RNA primer set for mitochondrial DNA as a quality check.
Wolbachia in individual field-collected Aedes albopictus from various collection sites
| Study sites | Types of habitat | Total female | Infected % | Infected female % (n) | Total Male | Infected % | Infected male % (n) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| wAlbA and wAlbB | wAlbA | wAlbB | Uninfected | wAlbA and wAlbB | wAlbA | wAlbB | Uninfected | ||||||
| Bukit Beruang, Malacca | Residential area | 13 | 100 | 100 (13) | 0 | 0 | 0 | 2 | 100 | 100 (2) | 0 | 0 | 0 |
| Besar Island, Malacca | Island | 26 | 100 | 100 (26) | 0 | 0 | 0 | 18 | 100 | 50 (9) | 0 | 50 (9) | 0 |
| Ketam Island, Selangor | Island | 22 | 100 | 100 (22) | 0 | 0 | 0 | 5 | 100 | 0 | 0 | 100 (5) | 0 |
| Carey Island, Selangor | Plantation | 4 | 100 | 100 (4) | 0 | 0 | 0 | 5 | 100 | 20 (1) | 0 | 80 (4) | 0 |
| Kajang, Selangor | Residential area | 12 | 100 | 100 (12) | 0 | 0 | 0 | 15 | 100 | 6.7 (1) | 0 | 93.3 (14) | 0 |
| Marang, Terengganu | Seashore | 17 | 100 | 100 (17) | 0 | 0 | 0 | 19 | 100 | 57.9 (11) | 0 | 42.1 (8) | 0 |
| Pangkor Island, Perak | Island | 32 | 93.7 | 87.5 (28) | 3.1 (1) | 3.1 (1) | 6.3 (2) | 22 | 86.4 | 54.6 (12) | 0 | 31.8 (7) | 13.6 (3) |
| Kuantan, Pahang | Plantation | 16 | 100 | 100 (16) | 0 | 0 | 0 | 16 | 87.5 | 87.5 (14) | 0 | 0 | 12.5 (2) |
| TOTAL | 142 | 98.6 | 97.2 (138) | 0.7 (1) | 0.7 (1) | 1.4 (2) | 102 | 95.1 | 49.0 (50) | 0 | 46.1 (47) | 4.9 (5) | |
Analysis of linearity and LOD determination.
The linear dynamic range for the multiplex RT-PCR assay was 100% at the range of 100 to 102 RNA copies per reaction but decreased to 88.8% at 10 copies. The LOD was set at 100 copies, which correspond to the mean Cq of 28. Samples with Cq value > 28 was scored as uninfected. Only mosquitoes that score above the LOD was reported to infer CHIKV infection and dissemination in midguts and salivary glands, respectively.
Wolbachia infection status in ovaries of mosquitoes used in the experimental oral infection study.
The presence of Wolbachia in females used in the experimental oral infection study was confirmed by screening the ovaries. It was noticed that ovaries for females were highly infected with at least 90% infection and above. There was no significant difference in the percentage of Wolbachia infection for all the time points for the three localities (P > 0.05, Fisher's exact test) (Figure 3).
The percentage of Wolbachia infection status in ovaries.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
The percentage of Wolbachia infection status in ovaries.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
The percentage of Wolbachia infection status in ovaries.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
Laboratory infection of Wolbachia infected (w+) and uninfected (w−) Ae. albopictus with CHIKV.
CHIKV midgut infections were observed for w+ and w− group for each time point, the midgut infection rate was consistent with percentage of at least 60% for w+ and 70% for w− for all localities (Figure 4A). There was no significant difference in the infection and dissemination rate between w+ and w− groups for all the time points tested for the three localities (P > 0.05, Fisher's exact test). It was noticed that CHIKV was detected in salivary glands as early as day 2 PI (Figure 4B).
The percentage of chikungunya virus (CHIKV) infection in (A) midguts and (B) salivary glands for Wolbachia infected (w+) and Wolbachia-free (w−) for all localities.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
The percentage of chikungunya virus (CHIKV) infection in (A) midguts and (B) salivary glands for Wolbachia infected (w+) and Wolbachia-free (w−) for all localities.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
The percentage of chikungunya virus (CHIKV) infection in (A) midguts and (B) salivary glands for Wolbachia infected (w+) and Wolbachia-free (w−) for all localities.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
The number of CHIKV genome copies in midguts was not significantly different between w+ and w− groups at any time point tested. CHIKV titer reached the peak as early as day 2 or day 3. For Besar Island, the peak titer for w+ and w− groups (median = 106.9 versus 107.9 viral copies/midgut, P = 0.05) achieved at day 3 PI. For Tenggol Island, the virus replication reached its peak at day 3 for w+ (median = 108.9 viral copies/midgut) and day 2 PI for w− (median = 108.3 viral copies/midgut), respectively. For mosquitoes sampled from Bandar Rinching, the viral copies were at the peak of viral infection for w+ and w− at day 3 PI (median = 107.8 versus 107.2 copies/midgut, P = 0.143) (Figure 5A).
Chikungunya virus (CHIKV) titer in (A) midguts and (B) salivary glands. Each symbol depicts a CHIKV load from individual organ. At each time point, 8–10 mosquitoes were killed for RNA extraction. Line indicates the median. Dotted line represents the limit of detection.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
Chikungunya virus (CHIKV) titer in (A) midguts and (B) salivary glands. Each symbol depicts a CHIKV load from individual organ. At each time point, 8–10 mosquitoes were killed for RNA extraction. Line indicates the median. Dotted line represents the limit of detection.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
Chikungunya virus (CHIKV) titer in (A) midguts and (B) salivary glands. Each symbol depicts a CHIKV load from individual organ. At each time point, 8–10 mosquitoes were killed for RNA extraction. Line indicates the median. Dotted line represents the limit of detection.
Citation: The American Society of Tropical Medicine and Hygiene 96, 1; 10.4269/ajtmh.16-0516
For salivary glands, the amount of CHIKV copies ranged from below the LOD (100 CHIKV copies) to 108 CHIKV copies in both w+ and w− groups. Although there was a variation in median CHIKV titer among the groups, these differences were not statistically significant at other time points, except for day 2 PI (Figure 5B). For Besar Island, CHIKV copy number of w+ group was almost one log lower than for w− group for salivary glands (medians = 101.8 versus 102.4, P < 0.05, Mann–Whitney U tests) (Figure 5B). Similarly, for Bandar Rinching, CHIKV copies in salivary glands for w+ was lower than w− group (median = 102.1 versus 103.3, P < 0.001, Mann–Whitney U tests) (Figure 5B).
The total CHIKV titer in different organs was compared between localities as shown in Table 2. The statistical analysis using Kruskal–Wallis test showed that total CHIKV titer in midguts were significantly different from at least one locality (P < 0.05). Subsequent post hoc analysis using the Bonferroni correction substantiated the difference. For w+, a significantly lower overall CHIKV titer in midguts was observed in Besar Island compared with Bandar Rinching and Tenggol Island. For w− group, a significantly higher overall CHIKV titer in midguts was observed in Tenggol Island compared with Bandar Rinching and Besar Island. No significant difference for CHIKV titer in salivary glands was observed among the three localities for w+ (P = 0.805) and w− (P = 0.431).
Median CHIKV copy number (IQR) of total CHIKV titer in midguts and salivary glands among localities for w+ and w− groups
| Locality | Sample size | CHIKV titer Median copy number (IQR) | ||||
|---|---|---|---|---|---|---|
| w+ | w− | |||||
| w+ | w− | Midguts | Salivary glands | Midguts | Salivary glands | |
| Besar Island, Malacca | 55 | 59 | 6.07 (5.21, 6.93)a* | 2.38 (1.63, 4.63)a | 7.02 (4.01, 7.80)a | 2.16 (1.22, 3.45)a |
| Tenggol Island, Terengganu | 58 | 60 | 7.44 (5.20, 8.09)b | 2.70 (1.45, 6.36)a | 7.83 (6.10, 8.53)b | 2.25 (1.44, 4.34)a |
| Bandar Rinching, Selangor | 60 | 57 | 7.01 (6.04, 7.69)b | 2.93 (1.73, 5.39)a | 6.98 (6.02, 7.50)a | 2.81 (1.28, 5.50)a |
Data are presented as median CHIKV copy number (IQR) and were compared among localities by Kruskal–Wallis test and Mann–Whitney U test for post hoc pairwise comparisons. CHIKV = chikungunya virus; IQR = interquartile range.
Median with different superscripts (i.e., a and b) within the same column indicates significant difference (P < 0.05).
Discussion
Our study on the distribution of Wolbachia in the field-collected Ae. albopictus showed a high infection rate of 98.6% in females and 95.1% in males. Females showed a common superinfection of wAlbA and wAlbB, occurred at a high prevalence of 97.2%. This is in accordance to other studies, showing superinfection percentage is common in field-collected Ae. albopictus infecting at least 96% of mosquitoes.35–39 Wolbachia are expected to rapidly spread to fixation once a Wolbachia infection enters a population.40,41 As females that carry both wAlbA and wAlbB strain can successfully mate with males that are either singly or superinfected, the superinfection of Wolbachia in females may explain the high fidelity of maternal transmission of Wolbachia of the mosquito species in the wild.42 The males were also most commonly superinfected with wAlbA and wAlbB at a percentage of 49.0% followed by 46.1% of wAlbB-only infection. No single infection of wAlbA was detected in males. Apart from the maternal transmission efficacy of Wolbachia, the geographical populations are reported to be strong predictors affecting Wolbachia infection rate and pattern.43 For example, a single-strain (wAlbA) infected population has been described in Koh Samui and Mauritius Islands that are geographically distinct from the superinfected populations.39,44,45 A study by Tortosa and others demonstrated that the density of wAlbA significantly decreased with age in male Ae. albopictus population in which a complete loss was observed within 5-day period postemergence.46 Therefore, this finding may serve as a plausible explanation for the lack of single wAlbA strain infection in the males despite having females that were superinfected with both wAlbA and wAlbB strain in the same population. The small percentage of uninfected samples may be due to Wolbachia leakage possibly related to the environmental factors such as high temperature and the effect of overcrowding during developmental stage of the larvae, which have been associated with reduced transmission of Wolbachia.47
The speculative assumption that native Wolbachia might affect the vectorial competence of the mosquitoes was further analyzed by investigating the percentage of CHIKV infection in midguts and dissemination to salivary glands. The artificial oral infection study was conducted in mosquitoes sampled from different geographical regions as genetic susceptibility of Ae. albopictus to CHIKV may vary by geography.48,49 In this study, removing Wolbachia did not induce any significant changes of mosquito response to infection by CHIKV. CHIKV was detected in midguts at a high rate of at least 60% and 70% for w+ and w−, respectively, regardless of the population tested. We measured CHIKV dissemination to secondary organs (salivary glands). CHIKV was detected as early as day 2 pi for both w+ and w−, suggesting a short extrinsic CHIKV incubation regardless the presence of Wolbachia. This finding is in line with other studies that demonstrated a short CHIKV incubation period of 2 days.50,51 We also demonstrated that the total CHIKV titer, but not infection susceptibility, is statistically affected by the geographical regions as previously reported.48
In this study, CHIKV can massively proliferate in midguts of w+ and w− groups. No difference in CHIKV titer was observed between w+ and w− groups for all time points except for day 2 PI for Besar Island and Bandar Rinching (salivary glands only). As CHIKV ingested by the mosquitoes must pass through the epithelium of the mosquito midgut before infecting salivary gland and other secondary organs, the occurrence of a midgut escape barrier in w+ group can be suggested, limiting the infection of salivary glands. However, as CHIKV inhibition effect was only observed at day 2 PI, it possibly explained a potentially weak midgut barrier caused by Wolbachia in its native hosts. Probably, the protection is caused by resource competition between Wolbachia and CHIKV in tissue in which they coexists. The presence of Wolbachia might limit the availability of resources that are important to ensure achievement of the viral cycle.23,52–54
The higher Wolbachia density confers a better protection toward viruses.55,56 However, the density of native Wolbachia in Ae. albopictus showed a high level of variation, whether it was from field- or laboratory-established populations.23,35 Additional studies to see correlation between virus concentration and Wolbachia density may provide a better insight to explain the inconsistency in the Wolbachia-mediated virus replication in the oral-challenge experiments in this study. However, the detection of the RNA genome only did not give definitive evidence of the viability of the virus. For example, Wong and others in the CHIKV oral-challenged in Ae. aegypti, wherein a persistent CHIKV RNA detection was reported in the mosquito eggs and adults progeny. However, it was not proven to be viable and infectious virus, omitting the possibility of the vertical transmission for CHIKV in Aedes sp.57 Nonetheless, we cannot exclude the limitation for using the quantitative PCR for virus detection as the RNA viral copy number can give an overestimation of infectious viral particles. Given the high sensitivity of real-time PCR in detecting the region encoding for E1 protein of CHIKV,57 even a slight contamination of virus nucleic acid on the surface of the organs (from tissue beside the organs) would have been sufficient to prime the RT-PCR reaction and hence yielding the overestimation of the infection percentage in the secondary organs including salivary glands.
Conclusion
Our results suggest a high prevalence of Wolbachia infection in the wild-caught Ae. albopictus. In accordance to other studies, the Ae. albopictus are naturally infected with wAlbA and wAlbB. Our data showed that the presence of Wolbachia do not pose any significant impact in the CHIKV infection in the midguts and dissemination to salivary glands in its native host, Ae. albopictus. The presence of Wolbachia does not interfere with the extensive CHIKV replication in midguts. Nonetheless, the native Wolbachia has a minimal effect on the CHIKV titer in the salivary glands, explaining why Ae. albopictus is a competent vector for CHIKV despite naturally infected with Wolbachia.
ACKNOWLEDGMENTS
We thank the Director-General of Health, Malaysia, and the Director, Institute for Medical Research (IMR), for permission to publish this study. We also thank Apandi Y and his team from Virology Unit, IMR, for providing and culturing the chikungunya virus, and Khairul Nizam MK from Molecular Diagnostic and Protein Unit for assisting us in the molecular work experiment. We acknowledge Nur Jannah J, Syakinah A, Chandru A, Azahari AH, Shakirudin N, Mahirah MN, and Khairul Asuad M, from Medical Entomology Unit, IMR, for their technical assistance in collecting, identifying, and dissecting the mosquitoes. This work formed a part of the graduate study (MSc) of the first author at University of Malaya, Kuala Lumpur, Malaysia.
- 1.↑
Gratz NG, 2004. Critical review of the vector status of Aedes albopictus. Med Vet Entomol 18: 215–227.
- 2.↑
Bonizzoni M, Gasperi G, Chen X, James AA, 2013. The invasive mosquito species Aedes albopictus: current knowledge and future perspectives. Trends Parasitol 29: 460–468.
- 3.↑
Delatte H, Paupy C, Dehecq JS, Thiria J, Failloux AB, Fontenille D, 2008. Aedes albopictus, vector of chikungunya and dengue viruses in Reunion Island: biology and control. Parasite 15: 3–13.
- 4.↑
Paupy C, Kassa F, Caron M, Nkoghe D, Leroy E, 2012. A chikungunya outbreak associated with the vector Aedes albopictus in remote villages of Gabon. Vector Borne Zoonotic Dis 12: 167–169.
- 5.↑
Arankalle VA, Shrivastava S, Cherian S, Gunjikar RS, Walimbe AM, Jadhav SM, Sudeep AB, Mishra AC, 2007. Genetic divergence of chikungunya viruses in India (1963–2006) with special reference to the 2005–2006 explosive epidemic. J Gen Virol 88: 1967–1976.
- 6.↑
Rezza G, Nicoletti L, Angelini R, Romi R, Finarelli AC, Panning M, Cordioli P, Fortuna C, Boros S, Magurano F, Silvi G, Angelini P, Dottori M, Ciufolini MG, Majori GC, Cassone A, 2007. Infection with chikungunya virus in Italy: an outbreak in a temperate region. Lancet 370: 1840–1846.
- 7.↑
Huang J, Yang C, Su C, Chang S, Cheng C, Yu S, Lin C, Shu P, 2009. Imported chikungunya virus strains, Taiwan, 2006–2009. Emerg Infect Dis 15: 1854–1856.
- 8.
Leo Y, Chow A, Tan L, Lye D, Lin L, Ng LC, 2008. Chikungunya outbreak, Singapore. Emerg Infect Dis 15: 836–837.
- 9.↑
Sam I, Chan Y, Chan S, Loong S, Chin H, Hooi P, Ganeswrie R, Abubakar S, 2009. Chikungunya virus of Asian and central/east African genotypes in Malaysia. J Clin Virol 46: 180–183.
- 10.↑
Rianthavorn P, Prianantathavorn K, Wuttirattanakowit N, Theamboonlers A, Poovorawan Y, 2010. An outbreak of chikungunya in southern Thailand from 2008 to 2009 caused by African strains with A226V mutation. Int J Infect Dis 14: 161–165.
- 11.↑
Gibney KB, Fischer M, Prince HE, Kramer LD, St George K, Kosoy OL, Laven JJ, Staples JE, 2011. Chikungunya fever in the United States: a fifteen year review of cases. Clin Infect Dis 52: e121–e126.
- 12.↑
AbuBakar S, Sam I-C, Wong P-F, Hooi P-S, Roslan N, MatRahim N, 2007. Reemergence of endemic chikungunya, Malaysia. Emerg Infect Dis J 13: 147.
- 13.↑
Noridah O, Paranthaman V, Nayar SK, Masliza M, Ranjit K, Norizah I, Chem YK, Mustafa B, Kumarasamy V, Chua KB, 2007. Outbreak of chikungunya due to virus of Central/East African genotype in Malaysia. Med J Malaysia 62: 323–328.
- 14.↑
Ttsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S, 2007. A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog 3: e201.
- 15.↑
de Lamballerie X, Leroy E, Charrel RN, Ttsetsarkin K, Higgs S, Gould EA, 2008. Chikungunya virus adapts to tiger mosquito via evolutionary convergence: a sign of things to come? Virol J 5: 1–4.
- 16.↑
Jeyaprakash A, Hoy MA, 2000. Long PCR improves Wolbachia DNA amplification: wsp sequences found in 76% of sixty-three arthropod species. Insect Mol Biol 9: 393–405.
- 17.↑
Hilgenboecker K, Hammerstein P, Schlattmann P, Telschow A, Werren JH, 2008. How many species are infected with Wolbachia?—a statistical analysis of current data. FEMS Microbiol Lett 281: 215–220.
- 18.↑
Breeuwer JAJ, Werren JH, 1990. Microorganisms associated with chromosome destruction and reproductive isolation between two insect species. Nature 346: 558–560.
- 19.↑
O'Neill SL, Karr TL, 1990. Bidirectional incompatibility between conspecific populations of Drosophila simulans. Nature 348: 178–180.
- 20.↑
Yeap HL, Mee P, Walker T, Weeks AR, O'Neill SL, Johnson P, Ritchie SA, Richardson KM, Doig C, Endersby NM, Hoffmann AA, 2011. Dynamics of the “popcorn” Wolbachia infection in outbred Aedes aegypti informs prospects for mosquito vector control. Genetics 187: 583–595.
- 21.↑
Joubert DA, Walker T, Carrington LB, De Bruyne JT, Kien DHT, Hoang NLT, Chau NVV, Iturbe-Ormaetxe I, Simmons CP, O'Neill SL, 2016. Establishment of a Wolbachia superinfection in Aedes aegypti mosquitoes as a potential approach for future resistance management. PLoS Pathog 12: e1005434.
- 22.↑
Skelton E, Rancès E, Frentiu FD, Kusmintarsih ES, Iturbe-Ormaetxe I, Caragata EP, Woolfit M, O'Neill SL, 2015. A native Wolbachia endosymbiont does not limit dengue virus infection in the mosquito Aedes notoscriptus (Diptera: Culicidae). J Med Entomol 53: 401–408.
- 23.↑
Mousson L, Zouache K, Arias-Goeta C, Raquin V, Mavingui P, Failloux AB, 2012. The native Wolbachia symbionts limit transmission of dengue virus in Aedes albopictus. PLoS Negl Trop Dis 6: 10.
- 24.↑
Glaser RL, Meola MA, 2010. The native Wolbachia endosymbionts of Drosophila melanogaster and Culex quinquefasciatus increase host resistance to West Nile virus infection. PLoS One 5: e11977.
- 25.
Hedges LM, Brownlie JC, O'Neill SL, Johnson KN, 2008. Wolbachia and virus protection in insects. Science 322: 702.
- 26.↑
Teixeira L, Ferreira A, Ashburner M, 2008. The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster. PLoS Biol 6: 2753–2763.
- 27.↑
Hoffmann AA, Iturbe-Ormaetxe I, Callahan AG, Phillips BL, Billington K, Axford JK, Montgomery B, Turley AP, O'Neill SL, 2014. Stability of the wMel Wolbachia infection following invasion into Aedes aegypti populations. PLoS Negl Trop Dis 8: e3115.
- 28.↑
Hoffmann AA, Montgomery BL, Popovici J, Iturbe-Ormaetxe I, Johnson PH, Muzzi F, Greenfield M, Durkan M, Leong YS, Dong Y, Cook H, Axford J, Callahan AG, Kenny N, Omodei C, McGraw EA, Ryan PA, Ritchie SA, Turelli M, O'Neill SL, 2011. Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission. Nature 476: 454–457.
- 29.↑
Zhang D, Lees RS, Xi Z, Gilles JRL, Bourtzis K, 2015. Combining the sterile insect technique with Wolbachia-based approaches: II—a safer approach to Aedes albopictus population suppression programmes, designed to minimize the consequences of inadvertent female release. PLoS One 10: e0135194.
- 30.↑
Bourtzis K, Dobson SL, Xi Z, Rasgon JL, Calvitti M, Moreira LA, Bossin HC, Moretti R, Baton LA, Hughes GL, Mavingui P, Gilles JRL, 2014. Harnessing mosquito—Wolbachia symbiosis for vector and disease control. Acta Trop 132: S150–S163.
- 31.↑
Ministry of Health. Guidelines on the Use of Ovitrap for Aedes Surveillance. Kuala Lumpur, Malaysia: MOH.
- 32.↑
Mahadevan S, Cheong WH, Hassan A, 1973. Vectors of Dengue and Dengue Haemorrhagic Fever in West Malaysia. Kuala Lumpur, Malaysia: Institute for Medical Research.
- 33.↑
Zhou W, Rousset F, O'Neill S, 1998. Phylogeny and PCR based classification of Wolbachia strains using wsp gene sequences. Proc Biol Sci 265: 509–515.
- 34.↑
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S, 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30: 2725–2729.
- 35.↑
Ahantarig A, Trinachartvanit W, Kittayapong P, 2008. Relative Wolbachia density of field-collected Aedes albopictus mosquitoes in Thailand. J Vector Ecol 33: 173–177.
- 36.
Armbruster P, Damsky WE, Giordano R, Birungi J, Munstermann LE, Conn JE, 2003. Infection of new- and old-world Aedes albopictus (Diptera: Culicidae) by the intracellular parasite Wolbachia: implications for host mitochondrial DNA evolution. J Med Entomol 40: 356–360.
- 37.
Dutton TJ, Sinkins SP, 2004. Strain-specific quantification of Wolbachia density in Aedes albopictus and effects of larval rearing conditions. Insect Mol Biol 13: 317–322.
- 38.
Kittayapong P, Baimai V, O'Neill SL, 2002. Field prevalence of Wolbachia in the mosquito vector Aedes albopictus. Am J Trop Med Hyg 66: 108–111.
- 39.↑
Sinkins SP, Braig HR, O'Neill SL, 1995. Wolbachia superinfections and the expression of cytoplasmic incompatibility. Proc Biol Sci 261: 325–330.
- 40.↑
Turelli M, Hoffmann AA, 1991. Rapid spread of an inherited incompatibility factor in California Drosophila. Nature 353: 440–442.
- 41.↑
Turelli M, Hoffmann AA, 1995. Cytoplasmic incompatibility in Drosophila simulans: dynamics and parameter estimates from natural populations. Genetics 140: 1319–1338.
- 42.↑
Kittayapong P, Baisley KJ, Sharpe RG, Baimai V, O'Neill SL, 2001. Maternal transmission efficiency of Wolbachia superinfections in Aedes albopictus populations in Thailand. Am J Trop Med Hyg 66: 103–107.
- 43.↑
Ahmed MZ, Araujo-Jnr EV, Welch JJ, Kawahara AY, 2015. Wolbachia in butterflies and moths: geographic structure in infection frequency. Front Zool 12: 16.
- 44.↑
Kambhampati S, Rai KS, Burgun SJ, 1993. Unidirectional cytoplasmic incompatibility in the mosquito, Aedes albopictus. Evolution 47: 673–677.
- 45.↑
Kittayapong P, Baisley KJ, Baimai V, 2000. Distribution and diversity of Wolbachia infections in southeast Asian mosquitoes (Diptera: Culicidae). J Med Entomol 37: 340–345.
- 46.↑
Tortosa P, Charlat S, Labbé P, Dehecq J-S, Barré H, Weill M, 2010. Wolbachia age-sex-specific density in Aedes albopictus: a host evolutionary response to cytoplasmic incompatibility? PLoS One 5: 9700.
- 47.↑
Wiwatanaratanabutr I, Kittayapong P, 2009. Effects of crowding and temperature on Wolbachia infection density among life cycle stages of Aedes albopictus. J Invertebr Pathol 102: 220–224.
- 48.↑
Tesh RB, Gubler DJ, Rosen L, 1976. Variation among geographic strains of Aedes albopictus in susceptibility to infection with chikungunya virus. Am J Trop Med Hyg 25: 326–335.
- 49.↑
Turell MJ, Beaman JR, Tammariello RF, 1992. Susceptibility of selected strains of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) to chikungunya virus. J Med Entomol 29: 49–53.
- 50.↑
Sam I-C, Loong S-K, Michael JC, Chua C-L, Wan Sulaiman WY, Vythilingam I, Chan S-Y, Chiam C-W, Yeong Y-S, AbuBakar S, Chan Y-F, 2012. Genotypic and phenotypic characterization of chikungunya virus of different genotypes from Malaysia. PLoS One 7: e50476.
- 51.↑
Vazeille M, Moutailler S, Coudrier D, Rousseaux C, Khun H, Huerre M, Thiria J, Dehecq J-S, Fontenille D, Schuffenecker I, Despres P, Failloux A-B, 2007. Two chikungunya isolates from the outbreak of La Reunion (Indian Ocean) exhibit different patterns of infection in the mosquito, Aedes albopictus. PLoS One 2: e1168.
- 52.↑
Moreira LA, Iturbe-Ormaetxe I, Jeffery JA, Lu G, Pyke AT, Hedges LM, Rocha BC, Hall-Mendelin S, Day A, Riegler M, Hugo LE, Johnson KN, Kay BH, McGraw EA, van den Hurk AF, Ryan PA, O'Neill SL, 2009. A Wolbachia symbiont in Aedes aegypti limits infection with dengue, chikungunya, and Plasmodium. Cell 139: 1268–1278.
- 53.
Walker T, Johnson PH, Moreira LA, Iturbe-Ormaetxe I, Frentiu FD, McMeniman CJ, Leong YS, Dong Y, Axford J, Kriesner P, Lloyd AL, Ritchie SA, O'Neill SL, Hoffmann AA, 2011. The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature 476: 450–453.
- 54.↑
Caragata EP, Rancès E, Hedges LM, Gofton AW, Johnson KN, O'Neill SL, McGraw EA, 2013. Dietary cholesterol modulates pathogen blocking by Wolbachia. PLoS Pathog 9: e1003459.
- 55.↑
Lu P, Bian G, Pan X, Xi Z, 2012. Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells. PLoS Negl Trop Dis 6: e1754.
- 56.↑
Osborne SE, Leong YS, O'Neill SL, Johnson KN, 2009. Variation in antiviral protection mediated by different Wolbachia strains in Drosophila simulans. PLoS Pathog 5: e1000656.
- 57.↑
Wong HV, Vythilingam I, Sulaiman WYW, Lulla A, Merits A, Chan YF, Sam I-C, 2016. Detection of persistent chikungunya virus RNA but not infectious virus in experimental vertical transmission in Aedes aegypti from Malaysia. Am J Trop Med Hyg 94: 182–186.