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1 !""#$%&'()*(+$,-./$!"#$%&"#'($)'(*'+$,-$.'#/.0$!12$,134-0$ 5+(6)+7#8#')$9:$;+<')+"$ 4$ HI#/(8""%&J$!?+)$%&#8$A%*+("+$J+&+6/$ C'/KJ(8I&A$$?9DEG3HLM$$ doi: /nature10355 Figure S1. Generation of stable wmel-transinfected A. aegypti lines resulting from embryo microinjection of wmel Wolbachia purified from a RML-12 cell line. A total of 2541 eggs were injected across 6 individual experiments using a hydrophilic membrane method of injection1. PCR screening using the IS5 element was carried out on females of the G0-G6 generations post-infection to determine the wmel infection status. Females from independently generated isofemale lines infected with the wmel strain were outcrossed to uninfected males until G4 at which time the lines were closed (matings only within the line). An outbred MGYP2.OUT line was established by backcrossing for 3 generations to the F1 progeny of wild-caught A. aegypti eggs from Cairns, Australia as outlined in Yeap et al.2. The F1 progeny from wild-caught eggs were used as control wildtype mosquitoes for comparison to outbred wmel-infected MGYP2.OUT mosquitoes. 1

2 Figure S2. Wolbachia wmel density in transinfected (G10) and outcrossed A. aegypti females. Density was determined by comparing the wsp (Wolbachia): rps17 (Aedes aegypti) gene ratios for 3-day-old female mosquitoes (n=10). The mean density (± s.e.m) of the wmel strain was not significantly different between transinfected and outcrossed lines (ANOVA; F (3,27) = 0.21, p=0.89). The density of the wmelpop-cla strain (red) in outcrossed PGYP1.OUT mosquitoes was significantly higher than the density of the wmel strain in outcrossed MGYP2.OUT mosquitoes (two sample t-test: t=5.94, df=14, p<0.001). 2

3 doi: /nature10355 Figure S3. Wolbachia infection in muscle and nervous tissue of Aedes aegypti female mosquitoes. Fluorescence in situ hybridization (FISH) of paraffin sections showing the localization of Wolbachia (red) in different tissues of 7-day-old female A. aegypti mosquitoes. Sections were hybridized with two Wolbachia-specific 16S rrna probes labeled with rhodamine. DNA is stained with DAPI (blue). A green GFP filter is used to provide contrast. 3

4 Figure S4. Fecundity (mean ± s.e.m) of wmel and wmelpop-cla infected mosquitoes in outbred mosquito lines under semi-field conditions. In the large semi-field cages, MGYP2.OUT (+wmel) and wildtype uninfected females were blood-fed on 5 different independent human volunteers (n=20-25 egg batches/volunteer). No significant differences in mean fecundity were observed between MGYP2.OUT and wildtype mosquitoes (two sample t-test: t=0.5804, df=245, p=0.5804). PGYP1.OUT (+wmelpop-cla) and wildtype uninfected females were also blood-fed on 4 different human volunteers in the semi-field cages (n=20-25 egg batches/volunteer) and the fecundity of PGYP1.OUT was significantly less than wildtype uninfected mosquitoes (two sample t-test: t=17.51, df=198, p<0.001). 4

5 Figure S5. Viability of Aedes aegypti eggs over time. 15 individual egg batches of Wolbachia-uninfected MGYP2.tet (black), wmel-infected MGYP2 (blue) and wmelpop-cla infected PGYP1 (red) were stored at 25 +/- 1 C and 85% relative humidity. Egg batches were hatched at 5, 12, 19 and 26 days post-oviposition by submersion in nutrient-infused deoxygenated water for 48 hours and the percentage of hatched eggs was scored. No significant differences were observed between the mean hatch rates of wmel-infected and uninfected lines at 5 (t=0.543, df=17, p=0.594), 12 (t=1.220, df=18, p=0.238), 19 (t=1.249, df=18, p=0.228) and 26 days (t=0.219, df=19, p=0.830) post oviposition. 5

6 Figure S6. Longevity of wmel-infected MGYP2.OUT and uninfected adult females. MGYP2.OUT (+wmel, blue line) and wildtype lines (-wmel, black line) were maintained at 25 +/- 1 C and 85% relative humidity with 25 females per cage (along with 25 males) with eight replicate cages per line. When pooled across replicates, longevity differed between lines by Cox regression (X 2 =12.00, df =1, p<0.001). When compared across replicate cages, there was a significant difference between lines for mean longevity (t=2.98, df=14, p=0.013) and a marginally non-significant difference for median longevity (t=2.05, df=14, p=0.060). 6

7 Figure S7. DENV-2 plaque forming units (PFUs) present in pooled saliva extracts. Mosquitoes were orally fed DENV-2 in an artificial blood meal (DENV-2 titer 1.5 x 10 7 PFU/mL) across three independent replicate experiments. Saliva was collected 14 days after blood feeding and pooled into groups of four for analysis to determine the presence of infectious virus in the saliva (n= saliva extracts/strain). *Both DENV-2 positive saliva pools from the MGYP2 line (each containing 2 PFUs) included saliva contributed from Wolbachia-uninfected females. 7

8 !""#$%$&"'"()"!"#$%&'"()&)$*)"+,"-.-$)!"!"#/+&0$12%$$%&'()*(+#'(,-.(/#!"#/+&0$12%$$%&'()*(+#,-.(/## 01#2&%&'()*(+#'(,-.(/# 01#2&%&'()*(+#,-.(/# 3*4*-.#56"7#,4/82%*4(/#9(.(-/(+#%&*4#)-:(/#%&#6#+-;/<# 3EFG#/)9((&%&:#4'#H577#.-9A-(#'49#%&'()I4&#/*-*2/<## 0-/./1('$&"'"()"$$ C2C-(#9(*29&(+#*4#)-:(<#*4#/%,2.-*(#4&:4%&:#9(.(-/(#C94:9-,# Figure S8. Semi-field cage invasion experiments. Initial starting frequency of Wolbachia-infected mosquitoes for both wmel and wmelpop-cla invasion experiments was 0.65, reflecting a release ratio of around 2 infected mosquitoes to 1 uninfected mosquito. A total of 1680 mosquitoes were introduced into the cage over a period of 6 days. Mosquitoes were provided access to a blood meal almost daily (at least 6 days per week). Eggs were collected on ovistrips every 3 days (starting from day 9) and after hatching, larvae were pooled and mixed and a sample of at least 100 larvae was used to assess Wolbachia infection frequency by PCR. The remaining larvae were reared to pupae and a sample of 60 female and 60 male pupae were returned to the cage every 3 days following the egg-collection schedule. Additional cohorts of Wolbachia-infected pupae (60 females and 60 males over a 6 day period) were regularly released in the cages (starting at day 7) over the course of the experiment to simulate an ongoing field release program. 8

9 Males MGYP2 MGYP2.OUT MGYP2.tet wildtype (+ wmel) (+ wmel) (- wmel) (- wmel) MGYP2 (+ wmel) ± ± 0.99 Females MGYP2.OUT (+ wmel) MGYP2.tet (- wmel) ± ± ± ± 0.95 Wildtype (- wmel) 0 ± ± 6.53 Table S1. Cytoplasmic incompatibility in wmel-infected A. aegypti mosquitoes. Crossing patterns resulting from matings between wmel-infected MGYP2 (+wmel) and Wolbachia-uninfected MGYP2.tet (-wmel) lines and between MGYP2.OUT (+wmel) and wildtype (-wmel) outbred lines. In the transinfected line crosses, values represent mean percent F1 embryo hatch ± s.e.m of individual gravid females after mass matings between 30 virgin female and male mosquitoes. In the outbred line crosses, values are based on mating between 10 virgin female and male mosquitoes. High F1 egg hatch rates are seen in crosses with wmel-infected females but no eggs hatch from crosses between uninfected females mated to wmel-infected males. 9

10 Sex Mosquito line Larval nutrition (mg food/larva/day) Development time (mean days ± s.e.m) MGYP ± 0.1 MGYP2.OUT ± 0.1 Females MGYP2.tet ± 0.1 wildtype ± 0.1 MGYP ± 0.6 MGYP2.tet ± 0.7 MGYP ± 0.1 MGYP2.OUT ± 0.1 Males MGYP2.tet ± 0.1 wildtype ± 0.1 MGYP ± 0.6 MGYP2.tet ± 0.5 Table S2. Larval development time (mean days ± s.e.m) of larvae reared under high (1 mg) and low (0.05 mg) larval nutritional levels (mg of food/larva/day) (n=50). Comparisons were made using two-sample t-tests between mosquito lines with the same genetic background. Under high nutrients, both male and female MGYP2 larvae developed faster than MGYP2.tet larvae counterparts (two-sample t-test, t=5.663, df=88, p<0.0001; t=6.968, df=88, p< respectively). Likewise, outcrossed MGYP2.OUT male and female larvae developed significantly faster than wildtype larvae counterparts (two-sample t-test: t=5.314, df=184, p<0.0001; t=5.506, df=157, p< respectively). Under low nutrients, no significant differences in the larval development time was seen for both male and female larvae of the MGYP2 and MGYP2.tet lines (two-sample t-test: t=1.836, df=158, p=0.068; t=2.537, df=143, p= respectively). 10

11 Modeling Wolbachia spread in semi-field cages To determine whether the increase in infection frequency in the cages was consistent with theoretical predictions, a deterministic age-structured model is used to describe the population in the cage, tracking daily cohorts of adult mosquitoes, categorized by sex and Wolbachia infection status, and their egg production. The model follows the basic structure of Rasgon et al. 3. We denote the numbers of non-infected and infected female adults in age class x on day t by U F (x,t) and I F (x,t), respectively, with the corresponding numbers of males written as U M (x,t) and I M (x,t). The age class label, x, takes integer values from 1 (newly emerged adults) through ω (age of oldest age class). We assume females mate once, one day after emergence, encountering their mate at random from the male population of mating age. Males are assumed to be ready to mate two days after emergence. The probability that a female mating on day t will mate with an infected male is given by, where each sum is taken over male age classes x that are of mating age. Females lay eggs according to an age-specific fecundity schedule that is dependent on infection status, as described by the functions F I (x) and F U (x). The reduction in daily fecundity due to Wolbachia infection means that F I (x) is smaller than F U (x). Based on the empirical data, we assume perfect maternal transmission, complete CI and that Wolbachia infection reduces the hatching probability of an infected egg by the factor q R. The frequency of Wolbachia infection for larvae that emerge from eggs laid on day t is given by 11

12 Here, sums are taken over age classes x for females of egg-laying age. The term t x + 1 that appears inside the function φ calculates the date on which the females of age x mated. Eggs laid in the cage are collected into three-day egg collections as specified by the schedule of the experiment. To calculate the infection frequency of larvae that emerge from each egg collection, we sum over numbers of larvae that emerge from eggs laid on the three relevant days. Numbers in the newly-emerged age class reflect the input and emergence of pupae. We assume that pupae emerge on the day after they are placed in the cage and that a fraction Φ of pupae emerge. (It is easy to see that the value of Φ has no effect on the Wolbachia frequencies predicted by our deterministic model: the dynamics of the model on the fractions of mosquitoes that are uninfected or infected. Because all individuals are introduced as pupae, changing Φ simply scales the total number of adult mosquitoes in the cage, leaving infected and uninfected fractions unchanged). Assuming a 50:50 sex ratio of emerging adults, we have the following equations for the numbers of newly emerged female adults, with a corresponding pair of equations for the numbers of males. Here, the numbers of infected and non-infected pupae introduced into the cage on day t are written as π I (t) and π U (t), respectively. These numbers can be split up according to their source (initial release, supplementary release, or return from eggs previously laid in the cage): π I (t) = π I init(t) + π I supp(t) + π I return(t) and π U (t) = π U init(t) + π U return(t). 12

13 Initial and supplementary release numbers are specified at the outset of the experiment, while the numbers π I return(t) and π U return(t) depend on the infection frequencies in pupae that develop from larvae hatched from eggs laid in the cage. The schedule of the experiment specifies the date on which pupae reared from a given egg collection are returned to the cage, so the return numbers are calculated as the product of the infection frequency in larvae that emerge from eggs laid in the relevant egg collection (see above) and number of pupae due to be returned. Notice that we make the assumption that the returned pupae are a representative sample of those that develop from eggs laid in a given egg collection (in particular, we assume that we need not distinguish between eggs laid on different days of an egg collection). Daily survival probabilities are dependent on age, sex and infection status; for infected and non-infected females we write these probabilities as p F,I (x) and p F,U (x), and for males we write p M,I (x) and p M,U (x). The daily changes in the numbers of females are given by the following recurrence relations:, for x = 1,, ω -1. A corresponding set of equations describes the changes in the numbers of males. For the wmelpop-cla experiments, the cage A model assumes a baseline 3% daily mortality for females, increasing to 10% for infected females after 14 days based on published information 2,4. Published mortality data are derived from lab studies; we assume that mosquitoes in the cage experience an additional background daily mortality (e.g. due to predation), set equal to 3% for cage A. Male survival is assumed to be 80% that of females. Eggs are assumed to be first laid by females in age class 6 (i.e. 5 days after emergence). We take fecundity to decline linearly with age, with a faster rate of decline for infected females, based on published results 3. With a relative 13

14 hatch rate of infected eggs, q R, set at 0.34, and all pupae emerging, these data provide a reasonable fit to the data from cage A (Fig 2b). For the cage B model, the additional background mortality is assumed to be 15% (higher than cage A due to gecko activity), calculated multiplicatively (all survival probabilities multiplied by 0.85). Other parameters are assumed to be identical to those above. In this case, there is a sharper increase in infection frequency as a consequence of the higher daily mortality rate, which matches the pattern seen in cage B (Fig2b). wmel imposes lower deleterious fitness effects and has a much lower effect on longevity. As a result, we assumed the same daily mortality for wmelinfected females as for uninfected females (i.e., a baseline 3% daily mortality), and that male survival was again 80% that of females. We assumed that fecundity of wmel-infected females followed a similar linear decline to that of uninfected females. Fitness effects (combining both a reduction in fecundity and reduced hatching of infected eggs) were set at 24%. Additional background mortalities were taken to equal 3% and 15%, as in the wmelpop- CLA simulations. The lower fitness costs associated with wmel leads to a faster predicted increase in infection frequency, in agreement with the observed data (Fig2a). 14

15 Supplementary references 1 Benedict, M. (2007) Microinjection Methods for Anopheles Embryos. Chapter 3: Specific Anopheles Techniques. MR4 Methods in Anopheles Research. 2 Yeap, H. L. et al. Dynamics of the popcorn Wolbachia infection in Aedes aegypti in an outbred background. Genetics 187, Rasgon, J. L. et al. (2003) Wolbachia-induced mortality as a mechanism to modulate pathogen transmission by vector arthropods. J Med Entomol 40, McMeniman, C. J. & O'Neill, S. L. A virulent Wolbachia infection decreases the viability of the dengue vector Aedes aegypti during periods of embryonic quiescence. PLoS Negl Trop Dis 4, e748, doi: /journal.pntd