Infection Incidence and Relative Density of the Bacteriophage WO-B in Aedes albopictus Mosquitoes from Fields in Thailand

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1 Curr Microbiol (2011) 62: DOI /s Infection Incidence and Relative Density of the Bacteriophage WO-B in Aedes albopictus Mosquitoes from Fields in Thailand Arunee Ahantarig Nopmanee Chauvatcharin Toon Ruang-areerate Visut Baimai Pattamaporn Kittayapong Received: 4 June 2010 / Accepted: 8 September 2010 / Published online: 28 October 2010 Ó Springer Science+Business Media, LLC 2010 Abstract We have used real-time quantitative PCR to measure, for the first time, the relative phage WO-B orf7 density and infection incidence in Aedes albopictus mosquitoes from fields in Thailand. Our results showed that the infection incidence of phage WO-B in this mosquito, sampled from geographically different places in Thailand, was 97.9%. Average relative densities of the offspring were different when collected from diverse parts and reared under the same conditions in the laboratory. Our results also revealed that geographical differences within Thailand did not influence the maternal transmission rate of bacteriophage WO-B. In addition, the orf7 loci might not be strictly associated with Wolbachia, because less than 100% of them were maternally inherited. This discovery does not support the hypothesis that bacteriophage WO-B is involved in Aedes albopictus cytoplasmic incompatibility. Whether this bacteriophage actually is involved in Wolbachiainduced cytoplasmic incompatibility in this mosquito thus A. Ahantarig N. Chauvatcharin T. Ruang-areerate V. Baimai P. Kittayapong (&) Center of Excellence for Vectors and Vector-Borne Diseases, Faculty of Science, Mahidol University at Salaya, Phutthamonthon 4 Road, Nakhon Pathom 73170, Thailand grpkt@mahidol.ac.th; pkittayapong@msn.com A. Ahantarig scaah@mahidol.ac.th A. Ahantarig V. Baimai P. Kittayapong Department of Biology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand T. Ruang-areerate Epidemiology Section, Research Division, Armed Forces Research Institute of Medical Sciences, Rajvithi Road, Bangkok 10400, Thailand needs further investigation, and additional densities of phage WO-B loci should be integrated. Introduction Aedes albopictus (skuse) is one of the significant mosquito vectors of dengue fever in many parts of the world [10 12]. All natural populations of A. albopictus that have been surveyed are infected with Wolbachia and most of them harbor a superinfection of two Wolbachia strains [20, 21] designated as walba and walbb (Wolbachia A & B supergroups). Wolbachia are capable of inducing a wide range of reproductive abnormalities in their hosts, including cytoplasmic incompatibility (CI) [14]. CI promotes the spread of Wolbachia through populations and, as a result, has been proposed as a gene-driving system for the distribution of disease-blocking transgenes through populations of mosquito vectors. The factors clearly accountable for CI are as yet unknown. The phylogeny of Wolbachia and incompatibility types showed no conformity. Therefore, there was the assumption that the genes responsible for Wolbachia incompatibility are reasoned by extra-chromosomal particles, such as plasmids or phages [8, 20]. Masui et al. [13] discovered a bacteriophage-like genetic element of Wolbachia, which was tentatively called bacteriophage WO. This bacterial virus has been tentatively proposed as a genetic candidate for inducing CI [6, 9, 19]. WO prophage sequences in Culex pipiens represent variable markers that now open ways for investigating the molecular basis of CI, as well as Wolbachia s effect on its hosts and the structure and dynamics of Wolbachia populations [5]. Although, there are several studies about phage WO orf7 in Culex mosquitoes [17], the phage orf7 locus

2 A. Ahantarig et al.: Infection Incidence and Relative Density of the Bacteriophage WO-B 817 alone cannot predict CI type in the Culex strains. Nevertheless, Bordenstein et al. [2] found an interesting result about tripartite association between bacteriophage, Wolbachia, and arthropods in which bacteriophage inversely associate with the densities and phenotype of an obligate intracellular bacterium and reproductive parasitism. In this study, using specific real-time quantitative PCR (RTQ-PCR), we determined the relative phage WO-B density and the infection incidence of field-caught, rather than laboratory-reared, A. albopictus mosquitoes in Thailand. Materials and Methods Mosquito Specimens The mosquitoes were collected from the field using light traps, animal-baited nets, and mosquito-landing catches. Populations of the mosquito A. albopictus were from five different regions (13 provinces) and three islands (south of Thailand). The identification of A. albopictus mosquitoes to species level was performed using the morphological keys of Buei [3] and Rattanarithikul and Panthusiri [15]. Female mosquitoes were allowed to lay eggs and subsequently were kept at -70 C. The mosquitoes were maintained in the insectary at Mahidol University s Center for Vectors and Vector-Borne Diseases at C temperature and 75% relative humidity. DNA Extraction DNA from mosquito samples was extracted using the crude boiling method of O Neill et al. [14]. Samples were homogenized in 100 ll of STE buffer (100 mm NaCl, 10 mm Tris HCl, 1 mm EDTA, and ph 8.0), heated at 95 C for 10 min, centrifuged, and then kept at -20 C. Polymerase Chain Reaction (PCR) Detection PCR amplification was done using 20 ll reaction mixture volumes to check for the presence of phage WO-B using WOorf7 primers: WOorf7F[5 0 -GAA ATG CTT GTT CAG CTA ATA GC-3 0 ] and WOorf7R [5 0 -ATA AAT TCT CCT ATT TTT TCT GGC A-3 0 ][13]. The PCR thermal profile was used as described in Chauvatcharin et al. [4]. Defensin primers (encoding the insect immunity of the mosquito) were used as a quality control for DNA extraction [16]. Real-time Quantitative PCR (RTQ-PCR) The relative densities of phage WO-B orf7 in mosquitoes were quantified by a real-time PCR-based method in an ABI PRISM Ò 7000 Sequence Detection System from Applied Biosystems. In this study, the amplification reaction was monitored using an SYBR green [4]. For Wolbachia copy quantitation, the primers used were GF and AR for walba, and GF and BR for walbb (GF, 5 0 -GGT TTT GCT GGT CAA GTA A; AR, 5 0 -GCA TCT TTG GTA ACT ACT TTT; and BR, 5 0 -GCT GTA AAGAAC GTT GAT C) [16]. SYBR Green was used to measure the amount of a single-copy gene (wsp) in all DNA samples [16]. Results Bacteriophage WO-B Infection Rate Aedes albopictus were caught in the field in various parts of Thailand. We used PCR with primers specific for the putative minor capsid protein (orf7) of bacteriophage WO- B to check for the infection incidence of phage WO-B in these mosquitoes. All samples were DNA quality checked with defensin primers. All negative samples for phage WO- B were Wolbachia positive with additional RTQ-PCR checked for phage WO density. The percentages of phage WO-B positive A. albopictus from geographically different regions and islands are reported. Global positioning system (GPS) of all provinces and islands are given in Table 1. Overall, the infection rate was 97.9% (93/95). Several provinces in each region gave the following results: north, 100% positive (20/20) (Chiang Mai, Chiang Rai, Lamphun, Lampang, and Phrae); northeast, 100% positive (15/15) (Khon Kaen, Ubon Ratchathani, and Surin); south, 100% positive (15/15) (Krabi, Satun, Phangnga, Surat Thani, and Songkhla); west, 100% positive (20/20) (Kanchanaburi); and east, 100% positive (15/15) (Chanthaburi). Mosquitoes sampled from islands (south of Thailand) had an overall infection rate of 80.0% positive (8/10) (Samui, Tao, and Phi-Phi). Offspring of mosquitoes from different sites were reared under the same conditions in the laboratory, and DNA extractions were performed. Negative results for phage WO-B were confirmed by RTQ-PCR and were PCR positive for Wolbachia. All adult mosquitoes from the F 1 generation were screened for the bacteriophage WO-B orf7. Not all were positive for phage WO-B (Table 2). Transmission varied even within the same part of Thailand. For example, in the northeast of Thailand, some samples were 100% transmitted (Khon Kaen 1), while others were 50% transmitted (Khon Kaen 2). Similar patterns were found in the west one F 0 in Kanchanaburi did not transmit WO phage to their offspring at all (Kanchanaburi 3). As with the F 0 generation, the DNA quality of all samples was checked with defensin primers. In addition, all negative WO samples were Wolbachia positive.

3 818 A. Ahantarig et al.: Infection Incidence and Relative Density of the Bacteriophage WO-B Table 1 GPS of all provinces and islands from different regions of Thailand Province GPS (UTM system) North Chiang Mai 47Q Chiang Rai 47Q Lamphun 47Q Lampang 47Q Phrae 47Q Northeast Khon Kaen 48Q Ubon Ratchathani 48P Surin 48P South Krabi 47P Satun 47N Phangnga 47P Surat Thani 47P Songkhla 47N West Kanchanaburi 47P East Chanthaburi 48P Island Samui 47P Tao 47P Phi-Phi 47N WO-B Density: F 0 versus F 1 The mosquito samples were from four provinces located in three geographically different regions of Thailand. All log relative phage WO densities were calculated from a phageto-bacteria ratio (normalizing the orf7 copy counts to the Wolbachia copy counts). Log relative phage WO-B densities within Wolbachia strains infecting F 1 A. albopictus mosquitoes were then determined. The log relative density of each F 0 individual was the average of triplicate experiments. Relative densities of phage WO orf7 were calculated from an average of at least four mosquitoes (F 1 ) from the same location and each one with triplicate wells. Log relative phage WO-B densities of F 0 and F 1 generation are shown in Fig. 1. Some F 0 mosquitoes (Songkhla and Kanchanaburi 2) had relatively lower densities of phage WO than those of their offspring, while those from Kanchanaburi 1, Khon Kaen, and Chanthaburi showed higher log relative phage WO density in F 0 than in F 1. The F 1 mosquitoes used in phage WO-B density determinations were adults, approximately 3 days old. Bacteriophage WO- B densities in A. albopictus mosquitoes (F 1 ) of the same age from different parts of Thailand were diverse, even though the mosquitoes were reared under the same conditions within the same laboratory. The log mean relative density of phage WO-B F 1 mosquitoes varied differently, especially among the F 1 mosquitoes of Kanchanaburi 1 and 2 (the same province), which showed very distinct WO Table 2 Phage WO-B maternal transmission rates in A. albopictus mosquitoes from different provinces in Thailand Province in Thailand Number of offspring positive for bacteriophage WO-B North 100% (5/5) Chiang Rai 5/5 Northeast 73.3% (11/15) Khon Kaen 1 7/7 Khon Kaen 2 4/8 South 77.8% (14/18) Phangnga 4/4 Krabi 4/4 Songkhla 6/10 West 57.6% (19/33) Kanchanaburi 1 15/15 Kanchanaburi 2 4/12 Kanchanaburi 3 0/6 East 66.7% (12/18) Chanthaburi 1 6/8 Chanthaburi 2 6/10 Fig. 1 Log relative density of phage WO-B from F 0 and F 1 A. albopictus mosquitoes from different provinces in Thailand. Age of F 0 mosquitoes caught in the field varied. Each F 1 bar represents the log relative density of mosquitoes (approximately 3 days old) from Songkhla (south) and Khon Kaen (northeast) with their associated standard errors. Chanthaburi (Chan) and Kanchanaburi (Kan) are in the east and west part of Thailand, respectively. All log relative bacteriophage WO orf7 densities was calculated from Wolbachia counts and phage WO counts (phage-to-bacteria ratio)

4 A. Ahantarig et al.: Infection Incidence and Relative Density of the Bacteriophage WO-B 819 relative densities when compared to each other: the significant difference being P \ at a = 0.05 (Fig. 1). Significant differences between F 0 and F 1 were found only in the provinces of Kan1 (P \ ), Kan2 (P \ ), and Chantaburi (P = 0.027). The differences were not significant in Songkhla (P = 0.365) and Khon Kaen (P = 0.358). Discussion In this study, overall infection incidence of bacteriophage WO-B of A. albopictus from fields was 97.9%. In all regions and islands the infection incidence for phage WO- B was 100%, except on one island (Tao Island) where two samples were negative (both of them were positive for Wolbachia). Since islands are separated from the mainland, they likely have vast climatic fluctuations (temperature and humidity) which may affect infection rates of phage WO- B. The infection incidence of phage WO-B in A. albopictus needs more investigation across greater geographical distances or different countries to see if ecological habitat does influence phage WO infection rate and density variations. From this study, geographically diverse places in Thailand did not have any influence on the maternal transmission rate of bacteriophage WO, as shown in Table 2. But host genetic background might be an important factor involved in the rate of maternal transmission and density. Our results showed that maternal transmission rates of bacteriophage WO-B differed among mosquitoes even within the same province (Table 2). The results from this study differed from those of Sanogo and Dobson [17] in that the orf7 loci were strictly associated with Wolbachia and were 100% maternally inherited in C. pipiens. Bacteriophage WO-B of A. albopictus in Thailand were not 100% maternally transmitted, so the orf7 loci and Wolbachia might not be strictly associated in this mosquito. The reasons might be: (i) the loss of WO orf7 in nature or in the laboratory, and/or (ii) WO orf7 copy is very rare and cannot be detected (We found 0 copy number of phage WO in F 1 of Kanchanaburi 3 before normalization with Wolbachia copy number). All field-caught mosquitoes in this study were Wolbachia positive. Three species of Wolbachia infected mosquitoes [Aedes (Stegomyia) sp., Hodgesia sp., and Armigeres flavus] were found to be negative for WO-B phage based upon orf7 PCR results, implying that the Wolbachia in these three mosquito species were not infected with WO-B phage [4]. In previous research, phages were also not detected in two other specific groups of Wolbachia: infecting nematodes and Trichogramma species [7, 18, 20]. Those findings support our present results in that orf7 or phage might not be necessarily strictly associated with Wolbachia bacteria in A. albopictus mosquitoes. Thus, the idea of the relation of bacteriophage WO-B in CI is less believable in this mosquito. Nevertheless, according to Gavotte et al. [7], phage WO could be the vector of genes involved in Wolbachia s effect; however, the frequent rearrangements occurring in Wolbachia genomes could rapidly lead to the transfer of these genes on the chromosome itself, thereby breaking down the association between phage and the Wolbachia effect. The recent discovery of a temperate phage WO-B of Wolbachia containing ankyrin-encoding genes and virulence factors has led to intensifying debate that bacteriophage induces CI [2]. Attractive issues to be investigated further are the influence of phage WO densities from various additional phage loci, such as ankyrin-encoding genes [22], as well as gp15 gene s potential involvement in CI [1] and the possibility that Wolbachia is involved in CI. Acknowledgments We are grateful for anonymous reviewer(s) for the advice to improve this article during the review process. We also thank Dr. John R. Milne for reviewing the manuscript; Drs. Ronald Morales Vargas, Supanee Hirunkanokpun, and Supat Wiwatanaratanabutr for their helpful suggestions; and Mr. Kitti Theinthong, Ms. Samnieng Theinthong, and Miss Nutchaya Klinpikul for their technical assistance. References 1. Ahantarig A, Khumthong R, Kittayapong P, Baimai V (2008) Relative densities of bacteriophage WO and Wolbachia of Aedes albopictus mosquito during development. Ann Microbiol 58: Bordenstein S, Marshall ML, Fry AJ, Kim U, Wernegreen JJ (2006) The tripartite associations between bacteriophage, Wolbachia, and arthropods. PLoS Pathogens 2:e43 3. Buei K (1983) Pictorial key to species. Adult mosquitoes in Thailand. Ministry of Public Health, Bangkok 4. Chauvatcharin N, Ahantarig A, Baimai V, Kittayapong P (2006) Bacteriophage WO-B and Wolbachia in natural mosquito hosts: infection incidence, transmission mode and relative density. Mol Ecol 15: Duron O, Fort P, Weill M (2006) Hypervariable prophage WO sequences describe an unexpected high number of Wolbachia variants in the mosquito Culex pipiens. Proc Biol Sci 273: Fujii Y, Kubo T, Ishikawa H, Sasaki T (2004) Isolation and characterization of the bacteriophage WO from Wolbachia, an arthropod endosymbiont. Biochem Biophys Res Commun 317: Gavotte L, Henri H, Stouthamer R, Charif D, Charlat S, Bouletreau M, Vavre F (2007) A survey of the bacteriophage WO in the endosymbiotic bacteria Wolbachia. 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