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Table of Contents
RESEARCH ARTICLE
Year : 2018  |  Volume : 55  |  Issue : 4  |  Page : 265-270

Detection and distribution of Wolbachia endobacteria in Culex quinquefasciatus populations (Diptera : Culicidae) from Metropolitan Manila, Philippines


1 Department of Civil and Environmental Engineering, Ehime University, Matsuyama, Japan; Biological Control Research Unit, Center for Natural Science and Environmental Research, De La Salle University, Taft Ave Manila, Philippines
2 Biology Department; Biological Control Research Unit, Center for Natural Science and Environmental Research, De La Salle University, Taft Ave Manila, Philippines
3 Department of Civil and Environmental Engineering, Ehime University, Matsuyama, Japan

Date of Submission10-Nov-2017
Date of Acceptance25-Jun-2018
Date of Web Publication18-Apr-2019

Correspondence Address:
Kozo Watanabe
Department of Civil and Environmental Engineering, Ehime University, Bunkyo-cho 3, Matsuyama, Ehime, 790-8577, Japan

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.256561

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  Abstract 

Background & objectives: Culex quinquefasciatus is a peridomestic mosquito known for its ability to transmit pathogenic diseases such as filariasis and Japanese encephalitis. The development and use of novel and innovative vector control measures such as the utilization of Wolbachia, along with the existing ones, are necessary to prevent the transmission of these diseases. Studies exploring the diversity of Wolbachia, particularly in Cx. quinquefasciatus are very limited in the Philippines. Thus, the aim of the study was to detect the presence, distribution, and phylogenetic relationship of Wolbachia infections in Cx. quinquefasciatus in Metropolitan Manila, Philippines.
Methods: Adult Cx. quinquefasciatus mosquitoes were collected using a commercially available light-trap from May 2014–January 2015. Based on their sampling grids (n = 51), the adult mosquito abdomens were pooled and subjected to Wolbachia surface protein (wsp) gene amplification assay. Five selected wsp-positive samples were then sequenced and further analyzed to infer their phylogenetic relationship with known Wolbachia strains.
Results: A total of 1090 adult Cx. quinquefasciatus mosquitoes were collected. Pooled abdomens (n = 53) were then sorted based on their sampling grids for subsequent screening of wsp gene. Wolbachia infection rate was 59% (31/53). These infections were located at 29 (57%) sampling grids, and were observed to be widely distributed in the study area. Phylogenetic analysis indicated that the sample sequences were Wolbachia pipientis isolated from known hosts, Cx. pipiens and Cx. quinquefasciatus belonging to supergroup B clade.
Interpretation & conclusion: The study was able to demonstrate the prevalence and distribution of Wolbachia in Cx. quinquefasciatus in Metropolitan Manila, Philippines. The findings of this study are geared towards proposing a vector control program that utilizes the potential of Wolbachia as a biological control agent in preventing the transmission of Culex-borne diseases.

Keywords: Culex quinquefasciatus; Metropolitan Manila; Philippines; Wolbachia


How to cite this article:
Carvajal TM, Capistrano JR, Hashimoto K, Go KD, Cruz MJ, Martinez MB, Tiopianco VP, Amalin DM, Watanabe K. Detection and distribution of Wolbachia endobacteria in Culex quinquefasciatus populations (Diptera : Culicidae) from Metropolitan Manila, Philippines. J Vector Borne Dis 2018;55:265-70

How to cite this URL:
Carvajal TM, Capistrano JR, Hashimoto K, Go KD, Cruz MJ, Martinez MB, Tiopianco VP, Amalin DM, Watanabe K. Detection and distribution of Wolbachia endobacteria in Culex quinquefasciatus populations (Diptera : Culicidae) from Metropolitan Manila, Philippines. J Vector Borne Dis [serial online] 2018 [cited 2019 Jul 15];55:265-70. Available from: http://www.jvbd.org/text.asp?2018/55/4/265/256561




  Introduction Top


Culex quinquefasciatus, a common household mosquito, has seen an unprecedented growth across the Philippines and worldwide[1],[2]. They are vectors of several diseases including filariasis and Japanese encephalitis[1],[3],[4]. Filariasis is endemic in most of the provinces in the Philippines and is a serious public health threat, while Japanese encephalitis puts 37% of the Philippines population at risk[5],[6]. Studies have shown that disease control services and surveillance in the Philippines are challenging, which contributes to the persistence of filariasis. There is also very little to no data on the epidemiology, social, and economic impact of filarial disease in the country[7]. Additionally, due to high insecticide resistance, control of the Cx. quinquefasciatus mosquito vector has become more difficult[4].

Wolbachia is an intracellular bacterial endosymbiont found in the reproductive tissues of arthropods and some filarial nematodes[8],[9],[10],[11],[12]. The prevalence of this endosymbiont has been recorded to vary from 15 to 76% in known invertebrates[13],[14] and it is estimated to infect 60-65% of known insect species[15]. The multiplication of the bacterium primarily relies on vertical transmission through maternal inheritance and causing diverse reproductive alterations in its host to maximize its transmission to the next generation[15],[16]. The effects of Wolbachia in manipulating its host's reproduction are considered ambiguous, which could be mutualistic or pathogenic, depending on Wolbachia's interaction with its host species. Examples of these effects are sperm-egg incompatibility, parthenogenesis, cytoplasmic incompatibility, and feminization[8],[17],[18]. Some studies have elucidated the other effects of Wolbachia to its hosts' biology, physiology, immunity, evolution, reproduction, and ecology[10], [19],[20]. As of to date, the most important application of this endosymbiont is its potential use as a bacterial biological control agent, specifically against mosquito-borne diseases[21],[22]. There are currently 28 known strains of Wolbachia in the world[8],[10],[11],[23] and so far eight major clades have been identified, six of which are detected in insects, arachnids, and crustaceans[24],[25].

In the Philippines, the detection and the phylogeny of this endosymbiont has been studied primarily in an agricultural setting such as predatory mites[26], plant hoppers[27],[28], ants[29], and whiteflies[30]. Culex pipiens has also been studied, emphasizing its characterization[31] and phylogeny[32]. However, no studies have investigated Wolbachia in Cx. quinquefasciatus in the Philippines. Thus, the present study aimed to detect Wolbachia endosymbionts in Cx. quinquefasciatus populations in Metropolitan Manila and infer its phylogenetic relationship using the Wolbachia surface protein (wsp) molecular marker. The results of the study will add to the growing literature of Wolbachia studies conducted in the Philippines. Furthermore, since the mosquitoes are known vectors that transmit periodic filariasis in the country, these findings may provide a basis for the bacteria's potential ability as a biological control in preventing the spread of the disease.


  Material & Methods Top


Study area, collection, identification and processing

Metropolitan Manila or the National Capital Region (NCR) of the Philippines is located on the eastern shore of Manila Bay in southwestern Luzon Island (14 °50′ N latitude, 121 °E longitude). It is considered to be a highly urbanized area, composed of 16 cities and one municipality. Initially, the entire region was divided into 2.5 by 2.5 km sampling grids. [Figure 1](a) shows the mosquito collection sites (n = 51) during the period from May 2014 to January 2015, where a total of 155 households (Average = 3.03 households per grid) were surveyed. A commercially available trap, MosquitoTrap® (Jocanima, Philippines), was installed in the outdoor premises of each household for 3 to 5 days to collect adult mosquito samples. Afterwards, the collected mosquito samples were sorted and identified as Cx. quinquefasciatus using the morphological keys from Becker et al[33]. Pre-processing for molecular analysis was done by separating the abdomen of each individual mosquito sample using a sterile blade and needle-pointed forceps. Culex quinquefasciatus abdomens were pooled together based on their sampling grids and were restricted only to a maximum of 23 abdomens per pool. Thus, a total of 53 pooled abdomen samples were used for subsequent molecular detection studies.
Figure 1: Map of Metropolitan Manila, Philippines divided into 2.5 × 2.5 km grids—(a) Grey circles indicate the sampling grids for Culex sp mosquitoes while blank grids indicate no sampling was done; and (b) Black circles indicate sampling grids positive for Wolbachia infection in Culex sp. mosquitoes. Large black circles with numbers indicate samples used for sequences analysis.

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DNA extraction and polymerase chain reaction

Total genomic DNA was extracted from the pooled mosquito abdomens by following the Qiagen DNeasy® Blood and Tissue kit protocol. To determine positive Wolbachia infection in the pooled mosquito abdomen samples, the wsp protein was used as a molecular marker, with the following primer sequences: wsp 81F (5′-TGG TCC AAT AAG TGA TGA AGA AAC-3′) and wsp 691R (5′-AAA AAT TAA ACG CTA CTC CA-3′). The 20 μl final reaction volume composed of 10 × buffer, 25 mM MgCl2, 10 mM of each dNTPs, 10 μM forward and reverse primers, and 5.0 U/μl of TaKaRa® Taq DNA polymerase (Takara Bio Inc., Japan). The PCR thermal profiles were as follows: One cycle of initial denaturation at 95 °C for 3 min, followed by 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min for 30 cycles, and final extension at 72 °C for 3 min[34]. DNA extracted from wMelPop strain, provided by Monash University in Australia, was used as a positive control in the study as it is one of the established and major strains of Wolbachia. The expected size of the target marker varies from 590 to 632 base pairs. This product size was checked through electrophoresis with a 1.5% agarose gel set at 100 V for 30 min and was observed under a Gel Doc™ XR+Gel Documentation System (Bio-Rad Laboratories, USA).

wsp gene sequencing and phylogenetic analysis

The study sequenced only five amplified wsp sample products based on the location of the sampling grid [Figure 1]b. The amplicons were sent for sequencing to the Eurofins Genomics—Tokyo, Japan. These sequences were then subjected to nucleotide basic local alignment search tool (BLAST) in order to confirm the similarity of the sample sequences with deposited wsp sequences in GenBank (www.ncbi.nlm.nih.gov/genbank/). Multiple sequence alignment was performed using Clustal W program along with other 14 known wsp sequences serving as both in-group and out-group [Table 1]. Among these, seven wsp sequences were from different host organisms[23], while the remaining included Cx. pipiens and Cx. quinquefasciatus wsp sequences from different country origins[35],[36],[37]. Unweighted pair group method with arithmetic mean (UPGMA) was used to infer the phylogenetic relationship. This analysis was performed in molecular evolutionary genetic analysis (MEGA) software[38] version 6. All sample sequences were deposited to GenBank with accession numbers (MH218808-MH218812).
Table 1: Description of Wolbachia strains used for phylogenetic analysis

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  Results Top


A total of 1090 Cx. quinquefasciatus adults were collected from households. The average collection was 6.98 and 21.24 per household and sampling grid, respectively. Out of the 53 pooled abdomen samples of Cx. quinquefasciatus, 31 (59%) were detected to be positive with Wolbachia infection based on wsp gene amplification assay. Positive infection was observed at 29 (57%) sampling grids [Figure 1]b and was seen to be widely distributed throughout the study area. Further analysis showed that the selected wsp-positive pooled samples were highly similar (>99% identity) to the deposited Wolbachia sequences in GenBank. Phylogenetic analyses revealed that the wsp sample sequences were identical to known Wolbachia sequences in Cx. pipiens and Cx. quinquefasciatus from different country origins belonging to supergroup B clade [Figure 2]. This indicates that the strain found in Metropolitan Manila is likely Wolbachia pipientis.
Figure 2: wsp phylogenetic tree showing the relationship of Wolbachia in Culex sp of Metropolitan Manila (• Culex wMM) with other Wolbachia strains from supergoup A and B clades using unweighted pair group method with arithmetic mean (UPGMA), the boostrap consensus tree inferred from 1000 replicates. A total of 515 positions were in the final dataset.
This analysis was achieved using MEGA 6.


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  Discussion Top


The study determined the wide geographic distribution of Wolbachia infections in Cx. quinquefasciatus, indicating that the endosymbiont is naturally found in this species of mosquitoes in the Philippines. Phylogenetic analysis further revealed that the selected wsp sample sequences are identical (100%) to known sequences of other global Cx. pipiens and Cx. quinquefasciatus populations. This elucidates the limitation of the wsp marker as a diagnostic tool for detecting Wolbachia infection in discriminating different strain types[39]. The marker has been compared to the antigen protein typing in screening pathogenic bacteria[40] where it is determined to be unsuitable for phylogenetic analysis because of its extensive recombination and strong diversifying selection[19],[41],[42]. However, intraspecific variation of Wolbachia among different populations of Cx. pipiens and Cx. quinquefasciatus mosquitoes worldwide has been observed to contain very low genetic diversity[36],[43],[44]. It was rationalized that this may be due to host-endosymbiont specificity[19] or recent infection of this endobacterium in the Culex population[43] which substantiates the observed identical wsp sequences of the study from different geographical locations, especially in a fine-scale study area.

The detection of the endosymbiont in two species of Culex mosquitoes, Cx. pipiens[31] and Cx. quinquefasciatus in the Philippines confirms the basis of widespread Wolbachia infection in mosquitoes found in the tropics[34]. Infection rates of this endobacterium vary in different Culex species across different regions. In Cx. quinque-fasciatus, the prevalence rates were 91.2% in south India[45]. Culex pipiens, on the other hand, had prevalence rates from 70–100% in Russia[46], Iran[36], California, USA[47], and China[48]. Other Culex species in Thailand such as Cx. gelidus had a prevalence rate of 54%, while no infection was found in Cx. tritaeniorhynchus[49]. The varied presence of the endosymbiont in different populations of Cx. quinquefasciatus and its related species can be due to several factors like exposure to antibiotics or heat treatment that attributed to its removal from the host[50],[51]. It has been reported that complete removal of the endosymbiont was achieved when reared at 32 to 34 °C for six generations from selected insect hosts (e.g. Tetranychus urticae, Urolepis rufipes)[52],[53]. It can be deduced that temperature may be an important factor in determining the frequency or removal of Wolbachia infections in the field populations. Notably, this goes the same with different mosquito species, since lower densities of Wolbachia were observed in Ae. albopictus reared at 37 °C as compared to 25 °C[54].

Wolbachia strains belonging to supergroup B clade are capable of cytoplasmic incompatibility[44],[47],[55], pathogen development inhibition[56], and transinfection[57],[58]. Wolbachia infections in Cx. quinquefasciatus were found with increased lifespan but with decreased reproductive fitness[59]. It has also been observed that Wolbachia in Cx. quinquefasciatus reduces the viral titre of the West Nile virus[56] and fialarial competence[57]. However, this reduction is not seen in Japanese encephalitis[58]. Further, studies are needed in order to understand this endosymbiont's pathophysiology and action mechanism to potentially utilize it as a better biological control agent against Culex- borne diseases. Since, Cx. quinquefasciatus is known to be a disease vector in the Philippines, understanding the phenotypic effects of Wolbachia strains to the host mosquito is essential for establishing it as an effective and efficient tool for biological control.


  Conclusion Top


This study demonstrated the presence of Wolbachia from different populations of Cx. quinquefasciatus using the wsp marker, where it is widely distributed throughout Metropolitan Manila. Furthermore, phylogenetic analysis showed that selected sample sequences belong to supergroup B clade of Wolbachia and are identical to Wolbachia pipientis found in both the mosquito hosts of Cx. pipiens and Cx. quinquefasciatus. The findings of the current study also add to the growing literature of Wolbachia studies done in the Philippines and could be used as reference towards exploring its potential as a biological control agent for Culex-borne infections (e.g. filariasis, Japanese encephalitis) which are endemic in the Philippines.

Conflict of interest

Authors declare no conflict of interest.


  Acknowledgements Top


The study was financially supported by the (a) Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (16H05750, 17H01624); (b) JSPS Bilateral Joint Research Projects; (c) Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT)—Research programme for promoting construction of the global environment information platform; Feasibility study of critical applications; and (d) Leading Academia in Marine and Environmental Pollution Research (LAMER)—Ehime University (Y29-1-8).



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1]



 

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Abstract
Introduction
Material & Methods
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Acknowledgements
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