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Table of Contents
Year : 2019  |  Volume : 56  |  Issue : 2  |  Page : 92-97

Pilot survey of mosquitoes (Diptera: Culicidae) from southeastern Georgia, USA for Wolbachia and Rickettsia felis (Rickettsiales: Rickettsiaceae)

1 Jiann-Ping Hsu College of Public Health, Georgia Southern University, Statesboro, Georgia, USA
2 Jiann-Ping Hsu College of Public Health, Georgia Southern University, Statesboro; Georgia Department of Public Health, Environmental Health Section, Atlanta, Georgia, USA

Date of Submission18-Jun-2018
Date of Acceptance15-Nov-2018
Date of Web Publication31-Jul-2019

Correspondence Address:
Marina E Eremeeva
Jiann-Ping Hsu College of Public Health, Georgia Southern University, 501 Forrest Drive, Statesboro, GA 30458
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.263714

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Background & objectives: Mosquito surveillance is one of the critical functions of local health departments, particularly in the context of outbreaks of severe mosquito-borne viral infections. Unfortunately, some viral and parasitic infections transmitted by mosquitoes, manifests non-specific clinical symptoms which may actually be of rickettsial etiology, including Rickettsia felis infections. This study tested the hypothesis that mosquitoes from southeastern Georgia, USA may be infected with Rickettsia felis and Wolbachia, an endosymbiotic bacterium of the order Rickettsiales.
Methods: Specimens of the five most common mosquito species occurring in the region were collected using gravid and light-traps and identified using morphological keys. Mosquitoes were then pooled by species, sex, trap and collection site and their DNA was extracted. Molecular methods were used to confirm mosquito identification, and presence of Wolbachia and R. felis.
Results: Wolbachia DNA was detected in 90.8% of the mosquito pools tested, which included 98% pools of Cx. quinquefasciatus Say (Diptera: Culicidae), 95% pools of Ae. albopictus Skuse (Diptera: Culicidae), and 66.7% of pools of Cx. pipiens complex. Samples of An. punctipennis Say (Diptera: Culicidae) and An. crucians Wiedemann (Diptera: Culicidae) were tested negative for Wolbachia DNA. Three genotypes of Wolbachia sp. belonging to Group A (1 type) and Group B (2 types) were identified. DNA of R. felis was not found in any pool of mosquitoes tested.
Interpretation & conclusions: This study provides a pilot data on the high presence of Wolbachia in Cx. quinque-fasciatus and Ae. albopictus mosquitoes prevalent in the study region. Whether the high prevalence of Wolbachia and its genetic diversity in mosquitoes affects the mosquitoes’ susceptibility to R. felis infection in Georgia will need further evaluation.

Keywords: BioB gene; Georgia; mosquito; Rickettsia; Wolbachia; 16S rRNA gene

How to cite this article:
Anderson ML, Rustin R C, Eremeeva ME. Pilot survey of mosquitoes (Diptera: Culicidae) from southeastern Georgia, USA for Wolbachia and Rickettsia felis (Rickettsiales: Rickettsiaceae). J Vector Borne Dis 2019;56:92-7

How to cite this URL:
Anderson ML, Rustin R C, Eremeeva ME. Pilot survey of mosquitoes (Diptera: Culicidae) from southeastern Georgia, USA for Wolbachia and Rickettsia felis (Rickettsiales: Rickettsiaceae). J Vector Borne Dis [serial online] 2019 [cited 2023 Mar 27];56:92-7. Available from: http://www.jvbd.org//text.asp?2019/56/2/92/263714

  Introduction Top

Mosquitoes are the most dominant group of bloodsucking ectoparasites responsible for transmission of numerous viral, bacterial, and parasitic diseases of humans and other vertebrates[1]. The State of Georgia, USA is endemic for many species of mosquitoes including Culex quinquefasciatus Say (Diptera: Culicidae), Aedes albopictus Skuse and Ae. aegypti Linnaeus[2]. Mosquito surveillance is one of the critical functions of state and local health departments, particularly in the context of recent or potential outbreaks of severe mosquito-borne viral infections, including West Nile virus, dengue, chi-kungunya, and Zika. It should be emphasized that some viral and parasitic infections manifest with several nonspecific clinical symptoms which may in fact be of rickettsial etiology, including R. felis infections[3],[4],[5]. Such situations are known in Mexico, Africa, and Sri Lanka; these areas are affected by many concurrent tropical diseases presenting with febrile syndrome[3],[4],[5]. Georgia, USA, is a state which was previously an epicenter of flea-borne rickettsiosis and where R. felis and cat fleas are still very abundant[6]; however, mosquitoes in the United States have not been screened for presence of R. felis. This situation is in contrast to recent reports that mosquitoes, including Anopheles, Aedes and Culex spp., from Gabon, Côte d’Ivoire and China are PCR positive for R. felis DNA[7],[8],[9]. Furthermore, Anopheles gambiae Giles (Diptera: Culicidae) has been shown to transmit R. felis in an experimental setting[10]; these findings implicate mosquitoes as potential unrecognized vectors for this pathogen.

Many natural populations of mosquitoes are also infected to various degrees with Wolbachia pipientis, a maternally inherited gram-negative endosymbiotic bac- terium of the order Rickettsiales[11]. Wolbachia are known for reproductive parasitism; manipulation of host reproduction gives Wolbachia a means of genetic drive and permits rapid spread through uninfected insect populations. Wolbachia infections in mosquitoes are not ubiquitous and are dependent on the strain, the host and other pathogens present. Naturally infected Ae. albopictus, a vector of dengue virus and Culex pipiens Linnaeus (vector of West Nile virus) typically have a low Wolbachia density, and seldom exhibit any sign of severe manipulation of populations by Wolbachia[12]. Wolbachia in Ae. aegypti inhibits dengue infection, and decreases vector competence as a result of the pathogen interference phenomenon[12]. Understanding how different mosquito-borne Wolbachia strain-host combinations interact with various pathogens and whether any derivative effects on the pathogens occur, has become a vital part in Wolbachia-based control of mosquito-transmitted diseases.

Recent microbiome studies reported a very low abundance (0.35%) of Wolbachia in larvae of Ae. albopictus and Cx. quinquefasciatus collected in Athens, Georgia[13]. Wolbachia has been also detected in other blood sucking ectoparasites collected across the State of Georgia (USA), including Amblyomma americanum Linnaeus (Akari: Ixodida)[14], and Ctenocephalides felis Bouché (Siphonap-tera) and C. canis Curtis which are the main vectors of R. felisX5. The purpose of this study was to examine infection rates with Wolbachia and R. felis in mosquitoes prevalent in southeastern Georgia, USA. The City of Statesboro is one of the new sentinel surveillance sites funded by the State of Georgia due to concerns about expansion of Zika virus.

  Material & Methods Top

Collection and identification

Mosquitoes were collected from four locations in Statesboro, Georgia (32.4488 °N, 81.7832 °W) using four gravid traps [model 1712, John W. Hock Company, Gainesville, Florida (FL), USA] and four CDC miniature light-traps (model 512, John W. Hock Company, Gainesville, FL) on August 11, 2016 as a part of the city trapping/ surveillance project. The trapping sites were primarily in residential areas of the city with flat to gentle sloping topography and with vegetation ranging from minimal canopy and typical household shrubbery, to sites adjacent to large suburban forests, a church and elementary school. One site was at the city’s public works facility adjacent to a large drainage ditch and suburban forest. All traps were set in the evening between 1730 and 1900 hrs and retrieved the next morning between 1000 and 1200 hrs.

On the day the traps were set, the weather was mostly cloudy with a low/high temperature of 22.8 to 31.7 °C; while on the next day it was fair with a temperature ranging from 23.3 to 33.3 °C. During three days prior to setting the traps, it rained at various intervals.

Mosquitoes were identified using morphological keys[16],[17]; and 2–11 mosquitoes were pooled based on species, sex, trap, and collection site. Heads were removed from each specimen to prevent inhibition of PCR amplification[18]. DNA of mosquitoes was extracted using DNeasy Blood and Tissue Kit [Qiagen, Valencia, California (CA)] and stored at 4 °C prior to testing. Identification of Cx. pipiens complex mosquitoes was confirmed by detecting a fragment length polymorphism in the second intron of the Ace-2 nuclear gene according to the protocol of Smith and Fonseca[19].

PCR detection of Rickettsia felis and Wolbachia

Detection of R. felis DNA was performed using a TaqMan assay targeting the species-specific BioB gene[20]. Each reaction was set up using 4 μl of DNA, 10 pmol (final amount per reaction) of each forward (RF_BioBF: 5’-ATGTTCGGGCTTCCGGTATG-3’) and reverse (RF_ BioR: 5’-CCGATTCAGCAGGTTCTTCAA-3’ primers, 5 pmol probe (5’-6-FAM-GCTGCGGCGGTATTTTAG- GAATGGG-TAMRA-3’), and Brilliant III Master Mix (Agilent, Santa Clara, CA). Sterile DNase and RNase free-water was used as a negative control for each reaction; recombinant plasmid containing an insert of the R. felis BioB gene fragment was used as a positive control. PCR amplification and subsequent analysis was performed using a BioRad CFX96 Instrument (BioRad, Hercules, CA).

Detection of Wolbachia DNA was performed using a TaqMan assay targeting the 16S rRNA gene of Wolbachia, which was developed for the purpose of this study. The forward (WN16S-F: 5’-CACAGAAGAAGTCCTGGC- TAAC-3’) and reverse (WN16S-R: 5’-CGCCCTTTAC- GCCCAATAA-3’) primers and probe (WN16S-probe: 5’-HEX-CGGTAATACGGAGAGGGCTAGCGTTA- BHQ1-3’) combinations were selected based on a consensus region identified from a multiple sequence alignment of 16S rRNA gene sequences of Wolbachia downloaded from NCBI GenBank and synthesized by Eurofin Genomics (Louisville, Kentucky). Each reaction was set up using 1.5 μl of DNA, 3.75 pmol of each forward and reverse primer and 1.5 pmol probe, and Brilliant III Master Mix (Agilent). After initial denaturation at 95 °C for 2 min, amplification consisted of 45 cycles of denaturation at 95 °C for 3 sec followed by annealing and elongation at 55 °C for 30 sec. Sterile DNase and RNase free-water was used as the negative control for each reaction, and DNA from two-lined spittlebug Prosapia bicinecta Say (Hemiptera: Cercopidae) containing Wolbachia DNA was used as the positive control.

Genotyping of Wolbachia

Wolbachia genotyping was performed using PCR amplification of wsp according to a previously described protocol[21] with slight modification. Primary amplification was performed using primers wsp81F (5’-TG-GTCCAATAAGTGATGAAGAAAC-3 ') and wsp691R (5’-AAAAATTAAACGCTACTCCA-3’) followed by two semi-nested PCR amplifications using genotype specific primers. Primers wsp328F (5’-CCAGCAGATAC- TATTGCG-3’) and wsp691R were used to amplify 363- bp fragment of Wolbachia genotype A. Primers 183F (5 '-AAGGAACCGAAGTTCATG-3 ') and 691R were used to amplify a 508-bp fragment of Wolbachia genotype B. All the PCR were set up using 2 μl of DNA, 40 pmol of each forward and reverse primers and Taq PCR Master Mix (Qiagen). PCR cycling conditions consisted of original denaturation for 5 min at 95 °C, followed by 35 cycles of 30 sec at 95 °C, 30 sec at 55 °C, 30 sec at 68 °C, and completed by final extension for 5 min at 72 °C. PCR products were separated on a 1% agarose gel for 30 min at 80 volts, stained with ethidium bromide and observed under UV-light.


Amplicons were purified using the Monarch® PCR and DNA Cleanup kit following the manufacturer’s recommendations (New England Biolabs, Ipswich, Massachusetts). DNA concentration in purified samples was determined using a Qubit 3.0 Fluorometer (Life Technologies Invitrogen, Carlsbad, CA). Samples were sequenced using Sanger sequencing (Clemson University Genomics Institute, Clemson, South Carolina). Sequences were analyzed for quality, edited, and contigs were assembled using Sequencher 5.4.1 (Gene Codes Corporations, Ann Arbor Michigan). Sequence identity was determined using the Basic Local Alignment Search Tool (BLAST) in GenBank. New sequences generated in this study were submitted to NCBI GenBank with accession numbers MG765532–MG765534.

Statistical analysis

Confidence intervals (CI) for minimal infection rate (MIR) and maximum likelihood estimate (MLE) for infection rate (IR) were calculated using CDC’s mosquito surveillance software for pooled data[22].

  Results Top

Overall, 552 mosquitoes were collected and identified using morphological criteria as Ae. albopictus (101), An. punctipennis Say (10), An. crucians Wiedemann (2), Cx. quinquefasciatus (407) and Cx. pipiens (32). PCR amplification of the ace-2 locus fragment from 47 pools of mosquitoes identified as Cx. quinquefasciatus produced a single fragment of 274-bp thus confirming their identification. There were 2 amplicons of 247-bp and 610-bp amplified from DNA of three mosquito pools which were morphologically identified as Cx. pipiens based on their lighter color and environmental conditions at the trapping site. Accordingly, a double-band profile was attributed to the presence of Cx. quinquefasciatus and Cx. pipiens hybrids; however, an accidental pooling of the two species can’t be excluded. For the purpose of this report those will be identified as Cx. pipiens complex since molecular identification was not done on individual specimens. Aedes albopictus and Cx. quinquefasciatus were present at all the four sites, An. punctipennis were collected at two locations, and Cx. pipiens complex mosquitoes were found only at one location.

A summary of the testing results is presented in [Table 1]. DNA of Wolbachia was detected in 90.8% (n = 76) of the mosquito pools tested, which included 46/47 (97.9%) pools of Cx. quinquefasciatus, 21/22 (95.5%) pools of Ae. albopictus, and 2/3 (66.7%) pools of Cx.pipi-ens complex. Samples of An. punctipennis and An. crucians tested negative for Wolbachia DNA. Estimated MIR per 100 mosquitoes ranged from 9.96 to 15.59% (95% CI). Bias corrected MLE for infection rate ranged from 36.94 to 67.93% (95% CI). There were no statistically significant differences in MIR between collections sites (p >0.05). DNA of R. felis was not detected in any of the 76 mosquito pools (comprising 552 mosquitoes) tested.
Table 1: Summary of TaqMan detection of R. felis and Wolbachia in mosquitoes collected

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Sequencing of the PCR amplicons generated using specific primer sets was used to determine the genetic types of Wolbachia infecting 11 Cx. quinquefasciatus and 12 Ae. albopictus mosquito pools. All the Cx. quinquefasciatus tested were positive for B genotype of Wolbachia. Nucleotide sequences of the amplicons generated from Cx. quinquefasciatus (MG765534) were most similar to the wPip wsp type (AF020060, AF020061) previously detected in Cx. pipiens from Tunisia and Cx. quinque-fasciatus from Gainesville, Florida. For Ae. albopictus, 9/12 pools were positive for A genotype and 10/12 pools for B genotype. Nucleotide sequences of the analyzed amplicons were most similar to the homologous wsp sequences of Wolbachia sp. WalbA and WalbB (GenBank: MG765532 and MG765533, respectively) both, previously identified in Ae. albopictus (GenBank: AF020058, AF020059) from Houston, Texas.

  Discussion Top

A total of five species of mosquitoes, endemic to Georgia, were examined in this study, viz. Cx. quinque-fasciatus, Cx. pipiens complex, Ae. albopictus, An. punc- tipennis and An. crucians. Culex quinquefasciatus was the most prevalent species; mosquitoes assigned to Cx. pipi-ens complex had a similar light appearance and were from an area surrounded with some standing water of clear appearance and less murky or eutrophic than other sites[23]. These specimens potentially could be the hybrids of Cx. quinquefasciatus and Cx. pipiens as demonstrated by ace- 1 restriction profile. The City of Statesboro, USA is located (32.4488 °N, 81.7832 °W) within the geographic limits of 30 and 40 °N, delineating the hybrid zone between these two species based on recent microsatellite studies[24],[25]. Hybrids of Cx. pipiens and Cx. quinquefasciatus mosquitoes are known to exhibit different host preference which may affect the patterns of pathogen transmission[19],[24],[25].

All the mosquitoes tested PCR negative for R. felis; however, most mosquito pools tested PCR positive for Wolbachia. The lack of detection of R. felis DNA in mosquitoes from Georgia is in contrast to findings previously reported from Côte d’Ivoire, Gabon and China[7],[8],[9]. In those studies, 1.3% (n = 77) An. gambiae from Côte d’Ivoire[8], 3.1% (n = 96) Ae. aegypti from Gabon[7], as well as 5.4% (n = 428) of An. sinensi (6.25%, n = 32) and Cx. pipiens pallens (5.5%, n = 396) from Jiangsu, China tested PCR positive for R. felis9. Furthermore, a closely related Rickettsia sp. has been detected in An. gambiae from these places[7],[8],[9], and recently Rickettsia sp. A12.2646, A12.2638 and A12.3271 have been identified using multiple locus sequencing from Mansonia uniformis, Cx. pipiens, and Ae. esoensis from Korea[26].

Since, all PCR controls for R. felis DNA performed as expected, it can be said that the negative PCR results indicate the absence of R. felis DNA in association with the mosquito pools tested in this study. This suggests that these mosquitoes, mostly trapped in urban locations, did not feed on animals which maintain R. felis rickettsemia. Virginia opossums (Didelphis virginiana) are considered to be the main reservoirs of R. felis in areas endemic for this pathogen in the USA. Opossums are common in Georgia and can be frequently seen in residential areas, and R. felis is highly prevalent in C. felis collected from Georgia[6]. Culex quinquefasciatus and Ae. albopictus were the most prevalent species examined in this study; Cx. quinquefasciatus is known for its preferential feeding on humans and dogs, but with only infrequent feeding on cats and opossums[27]. Aedes albopictus has been reported to exhibit an opportunistic host-feeding pattern with a predilection to feed on mammalian hosts, with most blood meals from humans, followed by dogs and cats, but only rarely on opossums[28],[29]. In contrast, studies performed in the neighbouring State of North Carolina, USA demonstrated frequent feeding of Cx. pipiens on opossum bloods in 55% of the samples tested[29]; however, Cx. pipiens was not prevalent in present collection. Similarly, Anopheles mosquitoes, reported R. felis-positive in other studies were not common among mosquitoes collected for this study.

The TaqMan assay used in this study for detection of R. felis is species-specific but it may amplify DNA of closely related R. asemboensis as determined by BLAST against its genome sequence. Detectability of “Candida-tus Rickettsia senegalensis” and other R. felis-like agents potentially present in the same areas can’t be predicted due to the uncultivated status of those rickettsiae, and the limited number of sequence targets available for these organisms in GenBank, mostly more conserved targets such as ompB, gltA or 17-kDa protein antigen gene. Although, it would be advantageous to have a broader detection or multiplex assay permitting simultaneous detection of R. felis and its nearest relatives, their prevalence is reported to be much lower when compared to that of R. felis[30],[31].

DNA of Wolbachia was detected in 66.7 to 97.9% pools of Culex mosquitoes and 95.5% of Ae. albopictus; however, DNA of Anopheles species tested negative for Wolbachia. The occurrence of Wolbachia A and B supergroups was identified based on sequencing of the wsp gene, the original typing system developed for Wolbachia[21]. Most recently multilocus sequence typing (MLST) based on sequencing of five genes has been developed and its usefulness has been demonstrated for discriminating between closely related strains, consequently, this typing provides more information for comparative genetics and molecular evolution of diverse genogroups of Wolbachia[32]. The relationships inferred using MLST and wsp sequences are complementary; furthermore, C. pipiens strains studied by Baldo et al[32] had identical MLST and wsp profiles suggesting that the use of wsp genotyping is adequate for the purpose of this pilot study. The MLST should be implemented as a part of future surveillance projects to confirm the preliminary findings using wsp and to obtain more information about the extent of the genetic diversity of Wolbachia in individual mosquitoes circulating in the region.

Wolbachia pipientis is a maternally inherited endo-symbiotic bacterium which is found in a large variety of insects and other arthropods, and filarial nematode populations[11]. Being found in different somatic and reproductive tissues of arthropods, Wolbachia can cause a variety of reproductive alterations, may affect the arthropod’s longevity, and can also exhibit an exclusion interference with pathogens in the arthropod[12]. These properties suggested the concept of utilizing Wolbachia as a biological vector-control strategy to reduce pathogen transmission by mosquitoes, particularly emerging arboviruses[33],[34]. Geographically divergent populations, biotypes and species of mosquitoes exhibit significant variation in infection with Wolbachia, ranging from complete absence to 80–100% prevalence[35],[36],[37]; this suggests that different mosquito populations may have different susceptibilities and/or protection against viruses or other pathogens associated with differences in Wolbachia carriage. Earlier studies conducted with laboratory maintained An. gambiae demonstrated horizontal transmission of R. felis. Rickettsia felis was visualized by immunofluorescence in salivary glands, in and around the gut, and in the ovaries of infected mosquitoes, and detected in faeces; however, no vertical transmission was observed[10]. This suggests an occurrence of possible exclusion interference effect (due to Wolbachia infection) in mosquitoes; however, this speculation needs further experimental confirmation and a better understanding of the genetic structure and diversity of Wolbachia genotypes present in mosquitoes, endemic to Georgia to determine if those mosquito strains of Wolbachia can inhibit acquisition of pathogens in the state.

  Conclusion Top

The result of the study indicates high prevalence of Wolbachia in Cx. quinquefasciatus and Ae. albopictus mosquitoes collected from Georgia, USA. Large sampling of under-represented mosquito species is needed to determine the regional presence of Wolbachia in these vectors. Further, studies are warranted to define the genetic diversity of circulating Wolbachia and to assess its variability within and among wild mosquito populations in Georgia.

Conflict of interest: The authors declare no conflict of interest. Ethical statement: Not applicable.

  Acknowledgements Top

Funding for this project was provided by 2013 and 2015 Georgia Southern University Faculty Research Awards to Marina Eremeeva. Matthew Anderson was supported by the 2016 American Society for Microbiology Undergraduate Research Fellowship, and Georgia Southern University’s College of Science and Mathematics COUR grant. The authors thank Dr Gregory A. Dasch (Centers for Disease Control and Prevention, Atlanta) for reviewing and providing helpful suggestions to improve this manuscript.

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