|Year : 2018 | Volume
| Issue : 4 | Page : 265-270
Detection and distribution of Wolbachia endobacteria in Culex quinquefasciatus populations (Diptera : Culicidae) from Metropolitan Manila, Philippines
Thaddeus M Carvajal1, Jayson Dale R Capistrano2, Kazuki Hashimoto3, Kristin Joyce D Go2, Maria Angeline Isabelle J Cruz2, Monique Johanne Lourdee B Martinez2, Vincent Stefano P Tiopianco2, Divina M Amalin2, Kozo Watanabe1
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 Submission||10-Nov-2017|
|Date of Acceptance||25-Jun-2018|
|Date of Web Publication||18-Apr-2019|
Department of Civil and Environmental Engineering, Ehime University, Bunkyo-cho 3, Matsuyama, Ehime, 790-8577, Japan
Source of Support: None, Conflict of Interest: None
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 Sep 20];55:265-70. Available from: http://www.jvbd.org/text.asp?2018/55/4/265/256561
| Introduction|| |
Culex quinquefasciatus, a common household mosquito, has seen an unprecedented growth across the Philippines and worldwide,. They are vectors of several diseases including filariasis and Japanese encephalitis,,. 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,. 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. Additionally, due to high insecticide resistance, control of the Cx. quinquefasciatus mosquito vector has become more difficult.
Wolbachia is an intracellular bacterial endosymbiont found in the reproductive tissues of arthropods and some filarial nematodes,,,,. The prevalence of this endosymbiont has been recorded to vary from 15 to 76% in known invertebrates, and it is estimated to infect 60-65% of known insect species. 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,. 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,,. Some studies have elucidated the other effects of Wolbachia to its hosts' biology, physiology, immunity, evolution, reproduction, and ecology, ,. 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,. There are currently 28 known strains of Wolbachia in the world,,, and so far eight major clades have been identified, six of which are detected in insects, arachnids, and crustaceans,.
In the Philippines, the detection and the phylogeny of this endosymbiont has been studied primarily in an agricultural setting such as predatory mites, plant hoppers,, ants, and whiteflies. Culex pipiens has also been studied, emphasizing its characterization and phylogeny. 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|| |
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. 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. 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, while the remaining included Cx. pipiens and Cx. quinquefasciatus wsp sequences from different country origins,,. 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 version 6. All sample sequences were deposited to GenBank with accession numbers (MH218808-MH218812).
|Table 1: Description of Wolbachia strains used for phylogenetic analysis|
Click here to view
| Results|| |
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.
Click here to view
| Discussion|| |
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. The marker has been compared to the antigen protein typing in screening pathogenic bacteria where it is determined to be unsuitable for phylogenetic analysis because of its extensive recombination and strong diversifying selection,,. 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,,. It was rationalized that this may be due to host-endosymbiont specificity or recent infection of this endobacterium in the Culex population 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 and Cx. quinquefasciatus in the Philippines confirms the basis of widespread Wolbachia infection in mosquitoes found in the tropics. 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. Culex pipiens, on the other hand, had prevalence rates from 70–100% in Russia, Iran, California, USA, and China. Other Culex species in Thailand such as Cx. gelidus had a prevalence rate of 54%, while no infection was found in Cx. tritaeniorhynchus. 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,. 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),. 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.
Wolbachia strains belonging to supergroup B clade are capable of cytoplasmic incompatibility,,, pathogen development inhibition, and transinfection,. Wolbachia infections in Cx. quinquefasciatus were found with increased lifespan but with decreased reproductive fitness. It has also been observed that Wolbachia in Cx. quinquefasciatus reduces the viral titre of the West Nile virus and fialarial competence. However, this reduction is not seen in Japanese encephalitis. 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|| |
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|| |
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).
| References|| |
Sarkar M, Bhattacharyya IM, Borkotoki A, Goswami D, Rabha B, Baruah, I, et al
. Insecticide resistance and detoxifying enzyme activity in the principal bancroftian filariasis vector, Culex quinquefasciatus
, in northeastern India. Med Vet Entomol
Al-Ali KH, El-Badry AA, Eassa AH, Al-Juhani AM, Al-Zubiany SF, Ibrahim EK. A study on Culex
species and Culex
transmitted diseases in Al-Madinah Al-Munawarah, Saudi Arabia. Parasitol United J
2008; 1(2): 101–8.
Corbel V, N'Guessan R, Brengues C, Chandre F, Djogbenou L, Martin T, et al
. Multiple insecticide resistance mechanisms in Anopheles gambiae
and Culex quinquefasciatus
from Benin, West Africa. Acta Trop
2007; 101(3): 207–16.
Galbo KR, Tabugo SR. Fluctuating asymmetry in the wings of Culex quinquefasciatus
(Say) (Diptera: Culicidae) from selected barangays in Iligan City, Philippines. AACL Bioflux
2014; 7(5): 357–64.
Kron, M, Walker E, Hernandez L, Torres E, Libranda-Ramirez B. Lymphatic filariasis in the Philippines. Parasitol Today
2000; 16(8): 329–33.
Werren JH. Biology of Wolbachia. Ann Rev Entomol
1997; 42(1): 587–609.
Tram U, Sullivan W. Role of delayed nuclear envelope breakdown and mitosis in Wolbachia-induced cytoplasmic incompatibility. Science
2002; 296(5570): 1124–6.
Serbus LR, Casper-Lindley C, Landmann F, Sullivan W. The genetics and cell biology of Wolbachia
-host interactions. Ann Rev Genet
Bandi C, Anderson TJ, Genchi C, Blaxter ML. Phylogeny of Wolbachia
in filarial nematodes. Proc R Soc Lond Biol Sci
Fischer P, Schmetz C, Bandi C, Bonow I, Mand S, Fischer K, et al
. Tunga penetrans: Molecular identification of Wolbachia
endobacteria and their recognition by antibodies against proteins of endobacteria from filarial parasites. Exp Parasitol
2002; 102(3): 201–11.
Werren JH, Windsor D, Guo L. Distribution of Wolbachia
among neotropical arthropods. Proc R Soc Lond Biol Sci
Jeyaprakash A, Hoy MA. Long PCR improves Wolbachia
DNA amplification: wsp
sequences found in 76% of sixty-three arthropod species. Insect Mol Biol
2000; 9(4): 393–405.
Rasgon JL. Wolbachia
induces male-specific mortality in the mosquito Culex pipiens
(LIN strain). PloS One
2012; 7(3): e30381.
Dobson Stephen L. Wolbachia
infections are distributed throughout insect somatic and germ line tissues. Insect Biochem Mol Biol
1999; 29(2): 153–60.
Bourtzis K, O'Neill S. Wolbachia
infections and arthropod reproduction. Bioscience
1998; 48(4): 287–93.
O'Neill SL, Hoffmann AA, Werren JH. Influential passengers: Inherited microorganisms and arthropod reproduction. Oxford (UK): Oxford University Press 1998; p. 232.
Werren JH, Baldo L, Clark ME. Wolbachia:
Master manipulators of invertebrate biology. Nat Rev Microbiol
2008; 6(10): 741–51.
Saridaki A, Bourtzis K. Wolbachia:
More than just a bug in insects genitals. Curr Opin Microbiol
2010; 13(1): 67–72.
Iturbe-Ormaetxe I, Walker T, O'Neill SL. Wolbachia
and the biological control of mosquito-borne disease. EMBO Rep
Dutra HL, dos Santos LM, Caragata EP, Silva JB, Villela DA, Maciel-de-Freitas R, et al
. From lab to field: The influence of urban landscapes on the invasive potential of Wolbachia
in Brazilian Aedes aegypti
mosquitoes. PLoS Negl Trop Dis
Zhou W, Rousset F, O'Neill S. Phylogeny and PCR-based classification of Wolbachia
strains using wsp
gene sequences. Proc R Soc London Biol Sci
1998; 265(1395): 509–15.
Lo N, Casiraghi M, Salati E, Bazzocchi C, Bandi C. How many Wolbachia
supergroups exist? Mol Biol Evol
2002; 19(3): 341–6.
Baldo L, Lo N, Werren JH. Mosaic nature of wsp (Wolbachia
surface protein). J Bacteriol
2005; 187(15): 5406–18.
Raros LA. Some new species records, discovery of males in two species and first report of Wolbachia
infection in predatory mites (Phytoseiidae, Acari) from the Philippines. Philipp Agric Sci
2005; 88(4): 431–9.
Qu LY, Lou YH, Fan HW, Ye YX, Huang HJ, Hu MQ, et al
. Two endosymbiotic bacteria, Wolbachia
, in the brown planthopper Nilaparvata lugens. Symbiosis
2013; 61(1): 47–53.
ShuPing L, Xin W, HongXing X, JiangWu T, XuSong Z, YaJun Y. Diversity of Wolbachia
in Anagrus nilaparvatae
(Hymenoptera: Mymaridae) analyzed using nested PCR-DGGE. Acta Entomologica Sinica
2011; 54(12): 1354–60.
Russell JA, Goldman-Huertas B, Moreau CS, Baldo L, Stahlhut JK, Werren JH, et al
. Specialization and geographic isolation among Wolbachia
symbionts from ants and lycaenid butterflies. Evolution
2009; 63(3): 624–40.
Jahan SM, Lee KY. Molecular variation of endosymbiotic bacteria Wolbachia
in Bemisia tabaci
and related whiteflies. Curr Res Agriculture Life Sci
2012; 30(2): 115–23.
Mahilum MM, Storch V, Becker N. Molecular and electron microscopic identification of Wolbachia
in Culex pipiens
complex populations from the Upper Rhine Valley, Germany, and Cebu City, Philippines. J Am Mosq Control Assoc
2003; 19(3): 206–10.
Duron O, Lagnel J, Raymond M, Bourtzis K, Fort P, Weill M. Transposable element polymorphism of Wolbachia
in the mosquito Culex pipiens:
Evidence of genetic diversity, superinfection and recombination. Mol Ecol
2005; 14(5): 1561–73.
Becker N, Petrić D, Boase C, Lane J, Zgomba M, Dahl C, et al
. Mosquitoes and their control. New York: Springer 2003. doi: 10.1007/978-3-540-92874-4.
Wiwatanaratanabutr I. Geographic distribution of Wolbachial
infections in mosquitoes from Thailand. J Invertebrate Pathol
2013; 114(3): 337–40.
Yildirim A, Inci A, Duzlu O, Onder Z, Ciloglu A. Detection and molecular characterization of the Wolbachia
endobacteria in the Culex pipiens
(Diptera: Culicidae) specimens collected from Kayseri province of Turkey. Ankara Universitesi Veteriner Fakultesi Dergisi
2013; 60(3): 189–94.
Karami M, Moosa-Kazemi SH, Oshaghi MA, Vatandoost H, Sedaghat MM, Rajabnia R, et al. Wolbachia
endobacteria in natural populations of Culex pipiens
of Iran and its phylogenetic congruence. J Arthropod-Borne Dis
Tsai KH, Lien JC, Huang CG, Wu WJ, Chen WJ. Molecular (sub) grouping of endosymbiont Wolbachia
infection among mosquitoes of Taiwan. J Med Entomol
2004; 41(4): 677–83.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol
Kageyama D, Narita S, Imamura T, Miyanoshita A. Detection and identification of Wolbachia
endosymbionts from laboratory stocks of stored-product insect pests and their parasitoids. J Stored Products Res
2010; 46(1): 13–9.
Pérez-Losada M, Viscidi RP, Demma JC, Zenilman J, Crandall KA. Population genetics of Neisseria gonorrhoeae
in a highprevalence community using a hypervariable outer membrane porB and 13 slowly evolving housekeeping genes. Mol Biol Evol
2005; 22(9): 1887–902.
Werren JH, Bartos JD. Recombination in Wolbachia. Curr Biol
Jiggins FM. The rate of recombination in Wolbachia
bacteria. Mol Biol Evol
Morais SA, Almeida FD, Suesdek L, Marrelli MT. Low genetic diversity in Wolbachia
-infected Culex quinquefasciatus
(Diptera: Culicidae) from Brazil and Argentina. Rev Inst Med Trop Sao Paulo
Atyame CM, Delsuc F, Pasteur N, Weill M, Duron O. Diversification of Wolbachia
endosymbiont in the Culex pipiens
mosquito. Mol Biol Evol
Sunish IP, Rajendran R, Paramasivan R, Dhananjeyan KJ, Tyagi BK. Wolbachia
endobacteria in a natural population of Culex quinquefasciatus
from filariasis endemic villages of south India and its phylogenetic implication. Trop Biomed
2011; 28(3): 569–76.
Vinogradova EB, Shaikevich EV, Ivanitsky AV. A study of the distribution of the Culex pipiens
complex (Insecta: Diptera: Culicidae) mosquitoes in the European part of Russia by molecular methods of identification. Comp Cytogenet
2007; 1(2): 129–38.
Rasgon JL, Scott TW. Wolbachia
and cytoplasmic incompatibility in the California Culex pipiens
mosquito species complex: Parameter estimates and infection dynamics in natural populations. Genetics
2003; 165(4): 2029–38.
Chen L, Zhu C, Zhang D. Naturally occurring incompatibilities between different Culex pipiens pallens
populations as the basis of potential mosquito control measures. PLoS Negl Trop Dis
2013; 7(1): e2030.
Tiawsirisup S, Sripatranusorn S, Oraveerakul K, Nuchprayoon S. Distribution of mosquito (Diptera: Culicidae) species and Wolbachia
(Rickettsiales: Rickettsiaceae) infections during the bird immigration season in Pathumthani province, central Thailand. Parasitol Res
2008; 102(4): 731.
Dutton TJ, Sinkins SP. Filarial susceptibility and effects of Wolbachia
in Aedes pseudoscutellaris
mosquitoes. Med Vet Entomol
2005; 19(1): 60–5.
Hermans PG, Hart CA, Trees AJ. In vitro
activity of antimicrobial agents against the endosymbiont Wolbachia pipientis. J Antimicrobial Chemother
2001; 47(6): 659–63,
Van Opijnen T, Breeuwer JA. High temperatures eliminate Wolbachia
, a cytoplasmic incompatibility inducing endosymbiont, from the two-spotted spider mite. Exp Appl Acarol
1999; 23(11): 871–81.
Kyei-Poku G, Benkel B, Goettel MS, Floate K. Elimination of Wolbachia
from Urolepis rufipes
(Ashmead) (Hymenoptera: Pteromalidae) with heat and antibiotic treatments: Implications for host reproduction. Biocontrol Sci Technol
2003; 13(3): 341–54.
Wiwatanaratanabutr I, Kittayapong P. Effects of temephos and temperature on Wolbachia
load and life history traits of Aedes albopictus. Med Vet Entomol
2006; 20(3): 300–7.
Baldo L, Bordenstein S, Wernegreen JJ, Werren JH. Widespread recombination throughout Wolbachia
genomes. Mol Biol Evol
2006; 23(2): 437–49.
Tsai KH, Huang CG, Wu WJ, Chuang CK, Lin CC, Chen WJ. Parallel infection of Japanese encephalitis virus and Wolbachia
within cells of mosquito salivary glands. J Med Entomol
2006; 43(4): 752–6.
Glaser RL, Meola MA. The native Wolbachia
endosymbionts of Drosophila melanogaster
and Culex quinquefasciatus
increase host resistance to West Nile virus infection. PloS One
2010; 5(8): e11977.
Kambris Z, Cook PE, Phuc HK, Sinkins SP. Immune activation by life-shortening Wolbachia
and reduced filarial competence in mosquitoes. Science
2009; 326(5949): 134–6.
de Almeida F, Moura AS, Cardoso AF, Winter CE, Bijovsky AT, Suesdek L. Effects of Wolbachia
on fitness of Culex quinque-fasciatus
(Diptera: Culicidae). Infect Genet Evol
[Figure 1], [Figure 2]