VARIATION IN ORAL SUSCEPTIBILITY TO DENGUE TYPE 2 VIRUS OF POPULATIONS OF AEDES AEGYPTI FROM THE ISLANDS OF TAHITI AND MOOREA, FRENCH POLYNESIA

Transcription

1 Am. J. Trop. Med. Hyg., 60(2), 999, pp Copyright 999 y The American Society of Tropical Medicine and Hygiene VARIATION IN ORAL SUSCEPTIBILITY TO DENGUE TYPE 2 VIRUS OF POPULATIONS OF AEDES AEGYPTI FROM THE ISLANDS OF TAHITI AND MOOREA, FRENCH POLYNESIA MARIE VAZEILLE-FALCOZ, LAURENCE MOUSSON, FRANÇOIS RODHAIN, ELIANE CHUNGUE, AND ANNA-BELLA FAILLOUX Unité d Ecologie des Systèmes Vectoriels, Institut Pasteur, Paris, France; Unité de Virologie, Institut Territorial de Recherches Médicales Louis Malardé, Papeete, Tahiti, French Polynesia Astract. Twenty three samples of Aedes aegypti populations from the islands of Tahiti and Moorea (French Polynesia) were tested for their oral susceptiility to dengue type 2 virus. The high infection rates otained suggest that the artificial feeding protocol used was more efficient than those previously descried. Statistical analysis of the results allowed us to define two distinct geographic areas on Tahiti with respect to the susceptiility of Ae. aegypti: the east coast, with homogeneous infection rates, and the west coast, with heterogeneous infection rates. No geographic differences could e demonstrated on Moorea. The possile mechanisms of this phenomenon are discussed in connection with recent findings on the variaility of susceptiility of Ae. aegypti to insecticides. The first documented outreak of dengue in Tahiti occurred in 944, as part of the Pacific-wide occurrence of the disease during World War II. The serotype responsile was dengue type. In the 960s, dengue virus type 3 was introduced soon after the opening of an international airport on Tahiti in 96. Thereafter, the comination of increased air traffic and uncontrolled uranization concomitant with population growth created ideal conditions for the introduction of dengue viruses and led to shortened interepidemic periods. All four serotypes were susequently found: dengue type in 975 and 988, dengue type 2 in 97 (with the first hemorrhagic manifestations reported in French Polynesia) 2, dengue type 3 in 969 and 989, and dengue type 4 in 979. After the epidemics of dengue type 4 in 979 and dengue type 3 in 989, transmission remained endemic at a low level until the arrival of a new serotype. 3 During the dengue type 2 epidemic of , 7,230 cases were reported y sentinel practitioners: 4,424 of these cases had a request for laoratory confirmation and 2,027 were confirmed and one death was reported. 4 Since no specific treatment and no dengue vaccine are yet availale, the prevention of dengue relies solely on control of vector populations. Two potential vectors of dengue viruses are present in French Polynesia: Aedes polynesiensis Marks and Ae. aegypti L. Although can replicate and transmit dengue viruses experimentally etween monkeys 5 and has een a vector for humans on islands where Ae. aegypti was not present, Ae. aegypti is of much greater epidemiologic importance on Tahiti. 6 Aedes aegypti was proaly introduced in French Polynesia at the end of the 9th century. It was first descried on Tahiti in the 950s. 7 Compared with, Ae. aegypti is not a very efficient vector of dengue viruses with a rather low relative susceptiility to oral infection. 8 Thus, only virus strains producing relatively high viremias in humans are transmissile y this mosquito. Rapid uranization of Tahiti has created conditions favoring reeding of Ae. aegypti in artificial containers near the vicinity of human populations. Aedes polynesiensis is not as closely in contact with humans and uses predominantly natural reeding sites such as coconut shells, tree holes, or cra holes. Little is known aout the role of the virus in the trans- 292 mission pattern and pathogenesis of dengue. Recent phylogenetic analysis ased on the variation of nucleotide sequence encoding for the envelope glycoprotein have shown variations in dengue viruses within each of the four serotypes. 9,0 In addition, variations in susceptiility to experimental oral infection with dengue viruses of geographic strains of Ae. aegypti and Ae. alopictus 2 was shown to e dose dependent and under genetic control. Although it has een demonstrated that different mosquito species or geographic colonized strains of the same species show variations in their susceptiility to experimental oral infection, little is known aout variaility among populations of the same mosquito species. The purpose of the present study was to investigate whether Ae. aegypti populations from two of the most uranized islands of French Polynesia, Tahiti and Moorea, showed variations in oral susceptiility to dengue type 2 virus. MATERIALS AND METHODS Mosquitoes. Twenty-three samples of Ae. aegypti were collected on Tahiti (49 30 W; 7 30 S) and Moorea (Figure and Tale ): nine in July and August 996 (no. 5; 3 6), and 4 in April 997 (no. 62; 723). The mosquitoes were collected either as larvae and/or pupae in reeding sites or as adults caught on human ait. These field-collected mosquitoes (F 0 generation) were maintained in the laoratory at 28 C with 80% relative humidity and a 6 hr:8 hr photoperiod. Adults were given 0% sucrose solution and females were allowed to feed on a restrained mouse to otain eggs. All F 0 adults were identified morphologically. For infection experiments, the entire egg atch was hatched and larvae were reared to the adult stage ( generation) in pans with tap water and yeast talets. Depending on the sample size, we tested either females or generation offspring from parents. The Paea strain of Ae. aegypti, which was provided y the Institut Louis Malardé (Tahiti, French Polynesia) and reared in Paris since 994, was used as a control of mosquito susceptiility. Virus. The dengue type 2 virus strain, provided y Leon Rosen (Institut Pasteur, Paris, France) was isolated in 974

2 SUSCEPTIBILITY OF AE. AEGYPTI TO DENGUE VIRUS 293 FIGURE. Locations of Aedes aegypti (numers) samples collected on Tahiti and Moorea. Inset, location of Tahiti and Moorea in the Society archipelago. from a human sera from Bangkok, Thailand. This virus had een passed only in different mosquito species (Toxorhynchites amoinensis, Ae. alopictus, and Ae. aegypti) y intrathoracic inoculation. 3 Viral stocks were produced y inoculating Ae. alopictus cells (C6/36 clone) 4 with triturated infected mosquitoes. The mosquito cells were maintained at 28 C on RPMI 640 medium supplemented with nonessential amino acids, penicillin, streptomycin, and 0% heatedinactivated (56 C for 30 min) fetal calf serum. The percentage of infected cells was monitored during the incuation period y the indirect fluorescent antiody (IFA) assay. 5 When 00% of the cells were infected, the supernatant fluid was collected, and the ph was adjusted to 7.5 with 0% sodium icaronate. The virus stock was divided into aliquots and stored at 80 C until used. Titration of the virus stock was carried out in Ae. aegypti (Paea strain) y inoculating serial dilutions of the supernatant intrathoracically. Mosquito infection was detected y an IFA assay on head squashes. Titers were calculated y the 50% endpoint method 6 and expressed as 50% mosquito infectious doses (MID 50 )/ml. Oral infection of mosquitoes. For each population, two infection assays were performed at a two-day interval when possile. For the nine populations collected on Tahiti in 996, individuals were tested in two assays performed etween Octoer and Decemer 996. For the 4 populations collected in 997 (seven for Tahiti and seven for Moorea), or individuals were tested in one or two assays, depending on the sample size, performed etween July and Septemer 997. The oral susceptiility of or females was tested y a feeding protocol adapted from Tardieux and others. 7 Briefly, 57-day-old females were deprived of sucrose solution 24 hr prior to the infectious meal and then allowed to feed for 20 min through a chicken skin memrane covering an apparatus containing the feeding mixture maintained at 37 C. The infectious meal consisted of two-thirds washed rait erythrocytes, one-third virus suspension, and ATP (as a phagostimulant) at a final concentration of M. Rait arterial lood was collected and erythrocytes were washed 2448 hr efore the infectious meal. Only fully engorged females were transferred to small cardoard containers and maintained at 28 C for 4 days. Surviving females were killed and tested for the presence of dengue virus y IFA assay on head squashes. Female mortality was also scored for each experiment. The Paea control strain was used to determine the optimal amount of virus to use in testing the field-derived populations. Statistical analysis. Variations in the proportions of surviving infected females at 4 days postinfection were compared using the rank column Fisher s exact test. The suprogram STRUC was used to compute an uniased estimate of the exact P value. 8

3 294 VAZEILLE-FALCOZ AND OTHERS TABLE Geographic origin of Aedes aegypti samples tested for experimental infection with dengue type 2 virus* No. Location Collection date Tahiti Moorea Papeete Faaa Punaauia Paea 2 Papara Vairao Tautira Taravao Faaone Hitiaa Tiarei Papenoo Mahina 2 Arue Pirae 2 Pirae 3 Vaiare Tiaia Opunohu Opunohu 2 Vaianae Manutea Manutea 2 * no other species found in the reeding site. 8/9/96 8/9/96 8/9/96 8/9/96 7/3/96 4/20/97 8/20/96 8/04/96 8/20/96 8/20/96 Generation tested Stage collected Other species found in the reeding site* Adult females Adult females Adult females and Culex quinquefasciatus and Cx. quinquefasciatus and Toxorhynchites amoinensis Cx. quinquefasciatus RESULTS Titer of the infectious meal. Prior to infecting field-derived populations, we determined the amount of virus needed to infect the Paea strain of Ae. aegypti. Serial dilutions of the viral stock were used in two different assays (Figure 2). The percentage of infected females detected 4 days after the infectious meal was dose dependent as shown y previous investigators, and ranged from 3% (for a lood meal with MID 50 /ml) to 00% (for a lood meal of MID 50 /ml). We used a feeding suspension with a final titer of 0 8 MID 50 /ml in all susequent comparative experiments to otain an average of 90% of infected females with the Paea control strain. Infection experiments with the Paea strain. The infection rates for the Paea control strain ranged from 64.7% to 00% (Figure 3). No significant difference was noted etween infection rates for the control strain in the first set of experiments (populations 5; 36) compared with the second set (populations 62; 723) of experiments (P 0.05). Nevertheless, one factor may have affected oral infection efficiency, namely, the time interval etween the collection of the rait lood and its use in feeding assays. In one case (infection performed in Octoer 996), we used erythrocytes collected and washed 72 hr efore the infectious meal. The infection rate of the Paea strain decreased to 9.35% (6 of 3) and the infection rate of the Papara fed at the same time (4 of 48) also was relatively low. This result led us to use erythrocytes collected no more than 48 hr efore the infectious meal. Mortality rates of infected mosquitoes. Cumulated mortality at 4 days after the infected meal ranged from 0% (Papara ) to 67.24% (Pirae 2). When compared with the mortality rate for the corresponding control using Fisher s exact test, eight experiments displayed significant differences (P 0.05): Papara, Taravao a, Hitiaa a, Arue, Pirae 2, Vaiare, Opunohu 2, and Vaianae a. High mortality rates were oserved among the experimental groups as well as among the control groups. Infection rates of Ae. aegypti samples. The infection rates for each sample, for each replicate, and for the Paea control strain are shown in Tale 2. Rates ranged from 70.50% (Manutea ) to 00% (Faaa a, Papara a, Taravao, and Pirae 3a). When compared with the infection rate for the corresponding control using Fisher s exact test, only four of the 39 assays exhiit significant differences (P 0.05): Vairao, Faaone, Tiaia a, and Manutea. The significant difference in susceptiility was in all instance related to the high level for the Paea control strain. When considering all experiments, homogeneity of infection rates was rejected (P 0 5 ). When analyzing each sample, homogeneity of infection rates etween replicates was only rejected for two samples from Tahiti: Punaauia (P ) and Arue (P 0.02). For these two samples, we took into account for further analysis the replicate with the higher sample size (Punaauia a and Arue ). Homogeneity of infection rates tested over all samples and replicates from one island was rejected when considering the

4 SUSCEPTIBILITY OF AE. AEGYPTI TO DENGUE VIRUS 295 FIGURE 2. Infection rates of the control Aedes aegypti Paea strain fed on increasing amounts of the dengue type 2 viral stock. percentage variation etween two independent infectious meals at each titer. MID 50 50% mosquito infectious dose. FIGURE 3. Infection rates of the control Aedes aegypti Paea strain orally infected with the dengue type 2 viral stock (titer of the meal 0 8 MID 50 (50% mosquito infectious doses)/ml. Assays : first set of experiments performed from Octoer to Decemer 996. Assays 2 23: second set of experiments performed from July to Septemer 997.

5 296 VAZEILLE-FALCOZ AND OTHERS TABLE 2 Infection rates of the 23 Aedes aegypti populations 4 days after oral infection with dengue type 2 virus* No. G Sample Replicate % of infected females (n) Assay Control P Tahiti Papeete a 87.0 (23) 83.3 (30) 60.0 (45) 77.4 (62) Faaa a 00 (2) 92. (38) 86.4 (22) 70.6 (34) Punaauia a 96.2 (78) 76.3 (59) 87. (70) 72. (43) Paea 2 a 8. (37) 77.0 (3) 64.7 (5) 79.0 (62) Papara a 00 (5) 86.7 (5) 94.7 (9) 00 (4) 6 Vairao a 90.2 (4) 97.5 (8) (42) 00 (7) Tautira a 79.7 (64) 88.9 (90) 85.9 (7) 86.2 (65) Taravao a 00 (26) 94.4 (72) Faaone a 90.2 (4) 97.5 (8) (79) 00 (7) Hitiaa a 83.9 (3) 92.2 (64) 0.29 Tiarei a 94. (34) 00 (65) Papenoo a 92.5 (40) 92.2 (64) 3 Mahina 2 a 82.6 (69) 77.3 (66) 87. (70) 72. (43) Arue a 69.6 (23) 9.9 (74) 82.6 (23) 86.7 (5) Pirae 2 a 85.5 (76) 84.2 (9) 73.3 (45) 77.4 (62) Pirae 3 a 00 (3) 92. (38) 86.7 (60) 70.6 (34) 0.09 Moorea 7 Vaiare a 97.9 (93) 00 (60) (98) 9.9 (37) 8 Tiaia a 9.3 (69) 00 (60) Opunohu a 94.3 (53) 8.8 () Opunohu 2 a 96.9 (98) 83.6 (55) 9.6 (83) 9.0 (67) Vaianae a 82.6 (23) 8.8 () 22 Manutea a 79.7 (74) 70.5 (39) 85.9 (7) 86.2 (65) Manutea 2 a 97.8 (92) 98.4 (64) 9.6 (83) 9.0 (67) * No. Location of the population shown in Figure ; G generation tested; n no. of females. Ae. aegypti Paea strain. P proaility of homogeneity from Fisher s exact test. Significant values (P 0.05) are in old. 6 Tahiti samples (P 0.03) and the seven Moorea samples (P 0 5 ). Since infection rates did not depend on the date of mosquito collection, all samples were considered to etter understand how the differences oserved in the infection rates were geographically structured. Tahiti, with an area of,04 km 2, is divided into a large section (Tahiti Nui) and a smaller section (Tahiti Iti) connected y the narrow isthmus of Taravao. For Tahiti, the samples were pooled in two groups (Figure 4): west coast (five samples) and east coast (nine samples), according to economic and ecologic considerations discussed later. Two samples, Papeete and Taravao, on the median line separating the two coasts, were excluded from the analysis (Figure 4). When tested for geographic differentiation, homogeneity of infection rates was accepted for the east coast group (P 0.384) and rejected for the west coast group (P 0.02) (Tale 3). When considering only samples from Tahiti Nui, homogeneity of infection rates remained differentiated for the west coast group (P 0.005) and nondifferentiated for the east coast group (P 0.392). For Moorea Island (Figure 4), the samples were pooled in two groups: north coast (three samples) and south coast (four samples). The south coast, where the port and the airport are located, is characterized y frequent daily connections (oth y air and sea) with the west coast of Tahiti. For oth groups, the homogeneity of infection rates was rejected: P 0.03 for the north coast group and P 0 5 for the south coast group (Tale 3). DISCUSSION The susceptiility of a mosquito strain to oral infection with dengue viruses generally is related to the numer of viral particles availale in the lood meal. With a feeding suspension containing 0 8 MID 50 /ml, we otained rates as high as 00%, whereas other investigators oserved lower

6 SUSCEPTIBILITY OF AE. AEGYPTI TO DENGUE VIRUS 297 FIGURE 4. Aedes aegypti population sudivisions on Tahiti and Moorea. The dotted area shows homogeneous field samples (numers) with respect to oral susceptiility with dengue type 2 virus. rates of infection with a similar amount of virus using different strains of Ae. aegypti and dengue type 2 virus.,7 A higher susceptiility to infection with Ae. aegypti from French Polynesia compared with other geographic areas could not e incriminated as the reason for our results ecause similar data were otained when we tested Ae. aegypti populations from French Guiana with the same virus strain. It is more likely related to the composition of the infectious meal. We used two volumes of erythrocytes for one volume TABLE 3 Proailities of homogeneity in infection rates with dengue type 2 virus among samples of Aedes aegypti according to their collection site on Tahiti and Moorea* Comparison N P Tahiti Whole island West coast East coast Tahiti Nui West coast East coast Moorea Whole island North coast South coast *N numer of samples and replicates; P proaility of homogeneity from Fisher s exact test. Significant values (P 0.05) are in old. of viral suspension, whereas other investigators,7 mixed one-third erythrocytes, one-third viral suspension, and onethird sucrose solution. We found that the quality of the erythrocytes also was a determinant of success in artificial oral infection. In addition, the quantity of erythrocytes in the meal could e an important factor. The high variaility oserved among replicates for the Paea strain may e due to the fact that this strain has not een selected for its oral susceptiility to dengue type 2 virus. Tardieux and others 9 compared isofemale lines originating from a strain of Ae. aegypti from French Polynesia (Fare strain) and demonstrated a highly significant difference for the oral susceptiility of these females for dengue virus type 2. Nevertheless, when testing the Paea control versus the samples, only four of 39 samples exhiited a significant difference, allowing us to validate our infection protocol. Oral susceptiility to dengue type 2 virus of Ae. aegypti from Tahiti and Moorea was highly differentiated when all samples and samples from the same island were considered. When considering Tahiti samples, two geographic areas were distinguishale, the west coast, with a high heterogeneity in infection rates, and the east coast, with a relative homogeneity of Ae. aegypti populations. Ecologic characteristics as well as particularities in human activity may explain the differentiation of these two areas. The west coast of Tahiti was originally covered y forests that were progressively replaced y haitations of an increasing population and modern development. With its wider coastal area and its drier

7 298 VAZEILLE-FALCOZ AND OTHERS climate, the west coast contains at least 75% of the inhaitants of Tahiti. Important economic infrastructures are also present. The hotels and the international airport provide the ideal means to introduce dengue viruses from elsewhere. In contrast, the east coast is windy and rainy with dense vegetation. This coast has suitale ecologic characteristics for the proliferation of, with aundant reeding places such as rock holes and tree holes. Except for two heavily populated localities of the east coast (Pirae and Arue) near Papeete, Ae. aegypti was rarely found, and then only in the vicinity of isolated haitations. During dengue outreaks, control measures against Ae. aegypti are undertaken on Tahiti y the Pulic Health Service using organophosphorous or pyrethroid insecticides. Control is more intensive in the vicinity of the airport than in more distant rural localities. Between dengue outreaks, the population uses commercial insecticides for personal protection. Consequently, insecticide pressure tends to e higher on the west coast, and resistance to insecticides in this area could e correlated with the intensity of their use. 20 As a result, the reduced numer of availale oviposition sites and their consequent greater heterogeneity may contriute to mosquito differentiation. 2 The heterogeneous distriution of infection rates in the mosquito populations from the west coast of Tahiti could e explained y genetically structured populations 22 selected y insecticides. This situation could lead to a selection of variants of dengue viruses introduced via the international airport. On the other hand, the homogeneous infection rates of Ae. aegypti on the east coast could e the result of lower selective pressures. Insecticide use is less extensive and re-invasions from untreated zones might tend to homogenize populations. In Moorea, 9 km west of Tahiti, differentiation in oral susceptiility to dengue type 2 virus was independent of the geographic location of mosquito samples, as for the west coast of Tahiti. Although the port and the airport are located on the southern side of the island, tourism activities are scattered all around the island. Furthermore, oth air and sea traffic are very intense etween Moorea, often considered a suur of Tahiti, and the west coast of Tahiti. This could explain the same pattern of heterogeneity in mosquito populations. Throughout the world, international airports are known to e important zones for the introduction and the dissemination of vector-orne diseases. This situation is particularly true in French Polynesia where viremic humans coming from dengue-infected areas could readily come in contact with a potential vector. This has led to the necessity to apply control measures continuously against Ae. aegypti in the vicinity of the airport and may have given rise to the differentiated and insecticide-resistant mosquito populations in this area. The variaility of the susceptiility of Ae. aegypti collected from distinct geographic locations to infection with dengue type 2 virus warrants further investigations to determine the genetic mechanisms underlying the efficiency of oral infection. Physical genetic mapping of Ae. aegypti ased on molecular markers such as restriction fragment length polymorphisms of microsatellites should facilitate the characterization of genes controlling the vector capacity traits. Acknowledgments: We thank Florence Fouque, Stéphane Laventure, and Tran Huu Hoang for help in collecting mosquitoes, and Nadia Monnier for rearing mosquitoes. We also thank Dr. Leon Rosen for a critical reading of the manuscript. Financial support: This work was partly supported y an Action Concertée des Instituts Pasteur (ACIP). Authors addresses: Marie Vazeille-Falcoz, Laurence Mousson, François Rodhain, and Anna-Bella Failloux, Unité d Ecologie des Systèmes Vectoriels, Institut Pasteur, 25 Rue du Docteur Roux, Paris Cédex 5, France; Eliane Chungue, Unité de Virologie, Institut Territorial de Recherches Médicales Louis Malardé, BP 30 Papeete, Tahiti, French Polynesia. REFERENCES. Rosen L, 958. Dengue antiodies in residents of the Society Islands, French Oceania. Am J Trop Med Hyg 7: Moreau JP, Rosen L, Saugrain J, Lagraulet J, 973. An epidemic of dengue on Tahiti associated with hemorrhagic manifestations. Am J Trop Med Hyg 22: Chungue E, Laudon F, Glaziou P, 993. Dengue and dengue haemorrhagic fever in French Polynesia Current situation. Trop Med 35: Chungue E, Deparis X, Murgue B, 998. Dengue in French Polynesia: major features, surveillance, molecular epidemiology and current situation. Pac Health Dialog 5: Rosen L, Rozeoom LE, Sweet BH, Sain AB, 954. The transmission of dengue y Aedes polynesiensis Marks. Am J Trop Med Hyg 3: Rosen L, 967. A recent outreak of dengue in French Polynesia. Jpn J Med Sci Biol 20 (suppl): Rosen L, 955. Oservations on the epidemiology of human filariaris in French Oceania. Am J Hyg 6: Rosen L, Rozeoom LE, Guler DJ, Lien JC, Chaniotis BN, 985. Comparative susceptiility of mosquito species and strains to oral and parenteral infection with dengue and Japanese encephalitis viruses. Am J Trop Med Hyg 34: Chungue E, Deuel V, Cassar O, Laille M, Martin PMV, 993. Molecular epidemiology of dengue 3 viruses and genetic relatedness among dengue 3 strains isolated from patients with mild or severe form of dengue fever in French Polynesia. J Gen Virol 74: Guler DJ, Trent DW, 994. Emergence of epidemic dengue/ dengue hemorrhagic fever as a pulic health prolem in the Americas. Infect Agents Dis 2: Guler DJ, Nalim S, Tan R, Saipan H, Sulianti Saroso J, 979. Variation in susceptiility to oral infection with dengue viruses among geographic strains of Aedes aegypti. Am J Trop Med Hyg 28: Guler DJ, Rosen L, 976. Variation among geographic strains of Aedes alopictus in susceptiility to infection with dengue viruses. Am J Trop Med Hyg 25: Rosen L, Guler DJ, 974. The use of mosquitoes to detect and propagate dengue viruses. Am J Trop Med Hyg 23: Igarashi A, 978. Isolation of a Singh s Aedes alopictus cell clone sensitive to dengue and chikungunya viruses. J Gen Virol 40: Kuerski TT, Rosen L, 977. A simple technique for the detection of dengue antigen in mosquitoes y immunofluorescence. Am J Trop Med Hyg 26: Reed LJ, Muench H, 938. A simple method of estimating fifty per cent endpoints. Am J Hyg 27: Tardieux I, Poupel O, Lapchin L, Rodhain F, 990. Variation among strains of Aedes aegypti in susceptiility to oral infection with dengue virus type 2. Am J Trop Med Hyg 43: Raymond M, Rousset F, 995. Genepop (version.2): population genetics software for exact tests and ecumenicism. J Hered 86: Tardieux I, Poupel O, Lapchin L, Rodhain F, 99. Analysis of inheritance of oral susceptiility of Aedes aegypti (Diptera:

8 SUSCEPTIBILITY OF AE. AEGYPTI TO DENGUE VIRUS 299 Culicidae) to dengue-2 virus using isofemales lines. J Med Entomol 28: Failloux A-B, Ung A, Raymond M, Pasteur N, 994. Insecticide susceptiility in mosquitoes (Diptera: Culicidae) from French Polynesia. J Med Entomol 3: Reiter P, Amador MA, Anderson RA, Clark GG, 995. Dispersal of Aedes aegypti in an uran area after lood feeding as demonstrated y ruidium eggs. Am J Trop Med Hyg 52: Failloux A-B, Darius H, Pasteur N, 995. Genetic differentiation of Aedes aegypti, the vector of dengue virus in French Polynesia. J Am Mosq Control Assoc :