Relative Wolbachia density of field-collected Aedes albopictus mosquitoes in Thailand

Transcription

1 Vol. 33, no. 1 Journal of Vector Ecology 173 Relative Wolbachia density of field-collected Aedes albopictus mosquitoes in Thailand Arunee Ahantarig 1,2, Wachareeporn Trinachartvanit 1, and Pattamaporn Kittayapong 1,2 1 Center for Vectors and Vector-Borne Diseases Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand 2 Department of Biology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Received 26 November 2007; Accepted 20 February 2008 abstract: Female Aedes albopictus mosquitoes from natural populations of different geographical regions of Thailand were collected and allowed to oviposit to determine relative Wolbachia A and Wolbachia B densities of their offspring (F 1 by using real-time quantitative PCR (RTQ-PCR. An important aspect of this work is that all Aedes albopictus mosquitoes were collected from the field. Twenty-seven offspring were from diverse areas of Thailand (Songkhla, Konkaen, Chantaburi, and Kanchanaburi. The range of relative Wolbabhia A density in F 1 mosquitoes was from to , whereas relative Wolbachia B densities ranged from 0 to These data are in contrast to those from a previous study that showed a very low amount (less than 0.10 of both relative Wolbachia density types for laboratory strains. The percent transmission of Wolbachia density from mother to each individual offspring cannot be predicted and was not related to the sex of the F 1. Obtaining confirmation for variations and unpredictable Wolbachia transmission load raises some concerns about using Wolbachia as a gene-driving system in nature for population replacement if Wolbachia density is involved in cytoplasmic incompatibility in this mosquito. Journal of Vector Ecology 33 (1: Keyword Index: Wolbachia, Aedes albopictus, real-time quantitative PCR. Introduction Aedes albopictus mosquitoes are important vectors of dengue fever, the disease that has become a major cause of death among people in various parts of the world (Knudsen 1995, Kambhampati and Rai 1991, Kambhampati et al Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods and are associated with a variety of reproductive alterations in their hosts, the best known being cytoplasmic incompatibility (CI (Kittayapong et al CI consists of sterility in cross matings, the crossing type being maternally inherited. It can be explained by the action of Wolbachia symbionts that are transmitted through the egg cytoplasm and leave an imprint on the sperm that prevents it from fertilizing unless it is rescued by the action of the same type of Wolbachia in the egg (Curtis and Sinkins, Most of the natural populations of Ae. albopictus harbor two types of Wolbachia, designated walba and walbb. Wolbachia-induced CI has been proposed as a potential mechanism to introduce and spread transmission-blocking genes into natural populations of insect vectors in an attempt to modify the vector competence of these populations (Sinkins et al The success of this long-term goal for disease control is critically dependent on the ability of Wolbachia to invade a host population and to establish a stable equilibrium prevalence within the target population that is high enough to have a significant impact on disease transmission (Sinkins et al. 1997, Turelli and Hoffmann, 1995, The exact mechanisms by which Wolbachia induce CI are still unknown. Several factors have been shown to modulate CI strength (i.e., egg hatchability, such as bacterial and host genotypes or bacterial density (reviewed in Weeks et al. 2002, and these factors may interact in complex ways. Wolbachia density is likely to influence the rate of maternal transmission and could also affect the penetrance of CI in incompatible crosses (Sinkins In contrast, the study by Duron et al. (2006 with Culex pipiens, suggested that Wolbachia load was not likely to be involved in CI for this species. The successful application of Wolbachia in insect vector control depends on the ability of the agent to successfully invade and maintain itself at a high frequency under field conditions (Kittayapong et al In addition, the success of Wolbachia as a gene-driving mechanism is also critically dependent on the efficiency of its maternal transmission under field conditions (Kittayapong et al In this study, the relative Wolbachia density in fieldcollected Ae. albopictus mosquitoes from four different geographical areas of Thailand were determined. Our results suggest a problematical issue for the use of Wolbachia as a gene-driving system for population replacement of this mosquito vector, if Wolbachia load is involved in CI mechanisms.

2 174 Journal of Vector Ecology June 2008 Materials and methods Mosquito collection and rearing Adult female mosquitoes from different geographical regions of Thailand were collected using light traps, animal-baited nets, and mosquito-landing catches. The morphological key of Buei (1983 and Rattanarithikul and Panthusiri (1994 were used to identify mosquitoes at the species level. All female mosquitoes were fed blood, then transferred to an individual vial to lay eggs and were subsequently stored at -70 C. Three-day-old F 1 adult mosquitoes were used in our experiments, reared at C and 75% relative humidity (RH in the insectary at the Mahidol University Center for Vectors and Vector-Borne Diseases. DNA extraction and polymerase chain reaction (PCR detection DNA was extracted using the crude boiling method of O Neill et al. (1992. All samples were ground and homogenized in 100 µl of STE buffer (100 mm NaCl, 10 mm Tris HCl, 1 mm EDTA, ph 8.0, heated at 95 C for 10 min, and centrifuged. PCR was performed to check for the presence of Wolbachia A and Wolbachia B. The PCR products were then diluted 500-fold and re-amplified by according to the protocol by Zhou et al. (1998. Defensin primers encoding immune peptides in the mosquito were used as a quality control for DNA extraction (Ruangareerate et al Real-time quantitative PCR (RTQ-PCR The relative densities of Wolbachia in mosquitoes were quantified by a real-time PCR-based method in an ABI PRISM 7000 Sequence Detection System from Applied Biosystems. In this study, SYBR green was used to monitor the amplification reaction. Each run consisted of a series of DNA standards prepared from plasmid DNA containing the wsp gene of either Wolbachia A or Wolbachia B from Wolbachia-infected Ae. albopictus mosquitoes (with 10 3 to copies of standard DNA as a template. Standard plasmid for Wolbachia A was constructed separately from that of Wolbachia B. Two and three replicated reactions were done for each standard and unknown, respectively. The PCR product of each gene was cloned into pgem- T vectors (Promega according to the manufacturer s recommendations and their sequences determined. The quality and concentration of all purified standard DNA was determined by recording a UV absorption spectrum at 260 nm with a spectrophotometer. The molar concentrations and the wsp gene copy number of all DNA were calculated after the determination of A 260. The signal curves of each standard and unknown measured in the same run were used for quantification and done automatically using computer software. Primers used were GF and AR for walba and GF and BR for walbb (GF, 5 -GGT TTT GCT GGT CAA GTA A; AR, 5 -GCA TCT TTG GTA ACT ACT TTT; BR, 5 - GCT GTA AAGAAC GTT GAT C (Ruang-areerate and Kittayapong RTQ-PCR cycling consisted of 95 o C for 15 min, followed by 45 cycles of 1 min at 94 o C, 1 min at 50 o C and finally, 1 min at 60 o C. Defensin primers were used to quantify mosquito gene copy numbers (Ruang-areerate Kittayapong Primers were designed specifically to the defensin of Ae. albopictus (Def-F, 5 -ATC ACT GGT GCT TAC CCA CAG G; Def-R, 5 -GAC GCA CAC CTT CTT GGA GTT G. SYBR Green was used to measure the amount of host cell numbers under the following conditions: 15 min at 95 C, then 45 cycles of 94 C for 1 min, 50 C for 1 min, and 72 C for 1 min. Wolbachia A, Wolbachia B and defensin copy numbers were analyzed separately with three different plasmid standards and the data were integrated to obtain relative bacterial densities. Results RTQ-PCR was used to assess the quantities of Wolbachia A and Wolbachia B in field-collected mosquitoes ( and F 1 from geographically different locations. Defensin copy numbers (host gene and Wolbachia copy numbers (bacterial gene were quantified and used to calculate relative Wolbachia A and Wolbachia B densities (bacteriato-host ratio. We constructed plasmid standards (two separated plasmids with Wolbachia A and Wolbachia B genes, including defensin plasmid standard gene. The target gene was quantified in the unknown sample of a mosquito host by a serial dilution of a standard in each RTQ-PCR run with a known amount of input copy number. Wolbachia bacterial density in each natural mosquito population was found to vary significantly. Defensin copy numbers of all mosquitoes ( -F 1 were in the range of 2.6 X 10 6 to 4.0 X 10 9 (Table 1 and 2. Twenty-seven F 1 mosquitoes from five females and four provinces of Thailand (Songkhla, Konkaen, Chantaburi, and Kanchanaburi were reared to 3-day-old adults (Table 1. All were RTQ-PCR quantified (triplicated experiments for each individual to determine relative walba and walbb densities. The results determined that some mosquitoes contained extreme walba loads whereas others had very high walbb densities. Bacterial densities of zero did not mean that the individuals had no Wolbachia copy numbers but that they contained the Wolbachia load in very small amounts when compared to host gene. This applied to all F 1 with relative Wolbachia densities of zero in this study. Interestingly, the densities of walba and walbb of mosquitoes (F 1 varied from each province. Even in Kanchanaburi (different females from the same province: -I, -II, there were large variations of walba densities in the F 1 generation (n=11: Table 1. For the Kanchanaburi -I, the offspring walba densities ranged from (bacteria-to-host ratio; Wolbachia A density was (Table 2. Wolbachia B density data ranged from (bacteria-to-host ratio; Wolbachia B density was (Table 2. For Ae. albopictus collected in Konkaen province ( (Table 2, walbb density was very high (32.6: bacteria-to-

3 Vol. 33, no. 1 Journal of Vector Ecology 175 Table 1. Relative Wolbachia A and Wolbachia B densities (bacteria-to host ratio of offspring (F 1 from geographically different regions of Thailand. Defensin copy numbers, which represented host genes, are also indicated for each individual mosquito. Mosquito province (F 1 Songkhla1 (south:f A density of each B density of each Songkhla2:F Songkhla3:F Defensin copy numbers of each 6.63x x x10 8 Songkhla4:M x10 8 Songkhla5:M x10 8 Konkaen1 (northeast:m x10 8 Konkaen2:M x10 8 Konkaen3:F x10 8 Konkaen4:F x10 8 Konkaen5:F x10 8 Chantaburi1 (east:f Chantaburi2:M Chantaburi3:M Chantaburi4:F Chantaburi5:M Chantaburi6:M Kanchanaburi-1 (west- ( -I:F Kanchanaburi-2:F Kanchanaburi-3:F Kanchanaburi-4:M x x x x x x x x x x10 8 Kanchanaburi-5:M x10 8 Kanchanaburi-1 (west- ( -II:M Kanchanaburi-2:F x x10 9 Kanchanaburi-3:F x10 9 Kanchanaburi-4:M x10 9 Kanchanaburi-5:M x10 9 Kanchanaburi-6:M x10 9

4 176 Journal of Vector Ecology June 2008 Table 2. Relative Wolbachia A and Wolbachia B densities (bacteria-to host ratio of mother ( from geographically different regions of Thailand. Defensin copy numbers represent host genes, and are also indicated in the table for each. Mosquito province A density of each B density of each Defensin copy numbers of each Songkhla x10 7 Konkaen x10 8 Chantaburi x10 8 Kanchanaburi- -I Kanchanaburi- -II x x10 9 host ratio and they can transmit this bacterial density to only some of their offspring (F 1 densities ranged from 0 to In contrast, Wolbachia A density in the F 1 of Konkaen were not so widely distributed ( as for Wolbachia B. Nonetheless, these numbers are considered very broad ranges compared to those of the laboratory strain. Wolbachia relative densities in mosquitoes from Songkhla (F 1, n=5 also differed in walba, which ranged from 6.55 to 9.34, but not as obvious as the F 1 mosquitoes from Konkaen. Surprisingly, mosquitoes from Songkhla ( contained very high bacterial densities of Wobachia B but their offspring had reduced walbb copy numbers. Among the bacterial densities of Ae. albopictus we determined from natural populations, transmission rates were not sex related as seen in Wolbachia A density of F 1 in Chantaburi 1 to 6, including high density variations in most F 1 mosquitoes from the other provinces. Discussion Three-day-old adult Ae. albopictus mosquitoes, reared in the laboratory under the same conditions but coming from different genetic backgrounds and environments when collected showed remarkable variations of Wolbachia A and Wolbachia B densities. The density variations were very high compared to a laboratory strain (Dutton and Sinkins An important aspect of this work is that all Ae. albopictus mosquitoes were from natural populations. Adult densities cannot be compared due to age variations when caught. Wolbachia transmission load from to F 1 was not equally distributed among all of the F 1 mosquitoes. Interestingly, maternally inherited Wolbachia transmission rates were not sex related (Table 1. That suggests bacterial densities may be transmitted to either male or female offspring randomly in unpredicted, high or low, amounts. Because of their capacity for population invasion and cytoplasmic replacement, Wolbachia have been suggested as a driving mechanism for spreading useful genes, such as those that may confer resistance to disease transmission. One essential property of any gene-driving system is that it can be used to achieve repeated population replacements. Even though the high fidelity with which the Ae. albopictus superinfection is passed to offspring in the field further supports the possible use of Wolbachia superinfections for this purpose (Kittayapong et al. 2002, Wolbachia transmission load variations in natural mosquitoes from this study suggests this needs to be reconsidered. In Cx. pipiens, Wolbachia density is not a critical factor for the CI rate and the bacterial dosage model proposed by Breeuwer and Werren (1993, in which CI strength correlates with relative infection levels in males and females of Drosophila simulan, cannot be applied to this species (Duron et al The data for Cx. pipiens differ from those obtained in other insect species, suggesting that hypotheses drawn from the Drosophila model cannot be generalized directly. Bacterial density appears to contribute relatively little to CI strength, compared to the major influence of the endosymbiont genotype (Duron et al In our study of field collected mosquitoes, some offspring obtained Wolbachia load from their mother in high amounts and others with very low copy numbers. If Wolbachia density is involved in CI mechanisms, it will be very difficult to use Wolbachia as a gene-drive system for population replacement because the degree of Wolbachia transmission of this mosquito in nature cannot be predicted. Even though this study only sampled a limited number of mosquitoes, reliable information can still be gained regarding the possible problems if Wolbachia density is involved in CI mechanisms in population replacement. The other vector control strategies, such as using an insecticide

5 Vol. 33, no. 1 Journal of Vector Ecology 177 resistance gene or bacteriophagewo in Wolbachia, might need to be considered more as alternative ways to study further to know more about the mechanisms of CI in order to control this vector in the field. Acknowledgments We thank Mr. Kitti Theinthong, Ms. Samnieng Theinthong, and Miss Nutchaya Klinpikul for their technical assistance. This work was supported by a Mahidol University Research Grant (SCBI-47-T-217. REFERENCES CITED Breeuwer, J.A.J. and J.H. Werren Cytoplasmic incompatibility and bacterial density in Nasonia vitripennis. Genetics 135: Buei K Pictorial Key to Species, Adult Mosquitoes in Thailand. Ministry of Public Health, Bangkok. Curtis, C.F. and S.P. Sinkins Wolbachia as a possible means of driving genes into populations. Parasitology 116 Suppl: S Duron, O., C. Bernard, S. Unal, A. Berthomieu, C. Berticat, and M. Weill Tracking factors modulating cytoplasmic incompatibilities in the mosquito Culex pipiens. Mol. Ecol. 15: Dutton T.J. and S.P. Sinkins Strain-specific quantification of Wolbachia density in Aedes albopictus and effects of larval rearing conditions. Insect Mol. Biol. 13: Kambhampati, S., W.C. Black, and K.S. Rai Geographic origin of the US and Brazilian Aedes albopictus inferred from allozyme analysis. Heredity 67: Kambhampati S. and K.S Rai Mitochondrial DNA variation within and among populations of the mosquito, Aedes albopictus. Genome 34: Kambhampati, S., K.S. Rai, and S.J. Burgun Unidirectional cytoplasmic incompatibility in the mosquito, Aedes albopictus. Evolution. 47: Kittayapong, P., K.J. Baisley, V. Baimai, and S.L. O Neill Distribution and diversity of Wolbachia infections in Southeast Asian mosquitoes (Diptera: Culicidae. J. Med. Entomol. 37: Kittayapong, P., P. Mongkalangoon, V. Baimai, and S.L. O Neill Host age effect and expression of cytoplasmic incompatibility in field populations of Wolbachia-superinfected Aedes albopictus. Heredity 88: Knudsen, A.B Global distribution and continuing spread of Aedes albopictus. Parasitologia 37: O Neill, S.L., R. Giordane, A.M.E. Colbert, T.L. Karr, and H.M. Robertsu S rrna phylogenetic analysis of the bacterial endosymbionts associated with CI in insects. Proc. Natl. Acad. Sci. USA 89: Rattanarithikul, R. and P. Panthusiri Illustrated Keys to the Medically Important Mosquitoes of Thailand, Wattana Panich Press, Bangkok. Ruang-areerate, T. and P. Kittayapong Wolbachia transfection in Aedes aegypti: A potential gene driver of dengue vectors. Proc. Natl. Acad. Sci. USA 103: Sinkins, S.P Wolbachia and cytoplasmic incompatibility in mosquitoes. Insect Biochem. Mol. Biol. 34: Sinkins, S.P., C.F. Curtis, and S.L. O Neill The potential application of inherited symbiont systems to pest control. In: S.L. O Neill, A.A Hoffmann, and J.H. Werren (eds. Influential Passengers. pp Oxford University Press, New York. Turelli. M. and A.A. Hoffmann Cytoplasmic incompatibility in Drosophila simulans: dynamics and parameter estimates from natural populations. Genetics. 140: Turelli, M. and A.A. Hoffmann Microbe-induced cytoplasmic incompatibility as a mechanism for introducing transgenes into arthropod populations. Insect Mol. Biol. 8: Weeks, A.R., K.T. Reynold, and A.A. Hoffman Wolbachia dynamics and host effects: what has (and has not been demonstrated? Trends Ecol. Evol. 17: Zhou, W., F. Rousset, and S.L. O Neill Phylogeny and PCR classification of Wolbachia strain using wsp gene sequences. Proc. R. Soc. Lond. B. 265: