The global expansion of Aedes albopictus has stimulated the development of environmental-friendly methods aiming at controlling disease transmission through the suppression of natural vector populations. Sterile male release programs are currently being deployed worldwide, and are challenged by the availability of an efficient sex separation that can be achieved mechanically at the pupal stage and/or by artificial intelligence at the adult stage, or through genetic sexing, which allows separating males and females at an early development stage. In this study, we combined the genetic sexing strain (GSS) previously established based on the linkage of dieldrin resistance to the male locus with a Wolbachia transinfected line. For this, we introduced either the wPip-I or the wPip-IV strain from Culex pipiens in an asymbiotic-Wolbachia-free Ae. albopictus line. We then measured the penetrance of CI and life history traits of both transinfected lines, selected the wPip-IV line, and combined it with the GSS. Population suppression experiments demonstrated a 90% reduction in population size and a 50% decrease in hatching rate. Presented results showed that such a combination has a high potential in terms of vector control but also highlighted associated fitness costs, which should be reduced before large-scale field assay.

Crossing experiments and effect of male age on CI intensity (data file "CI expression of Wolbachia transinfected lines in Aedes albopictus" and "Effect of male age on CI intensity"

To evaluate CI penetrance in both transinfected lines, we performed reciprocal en masse crosses using two laboratory lines (S-Run and asymbiotic). We then confirmed the CI pattern observed crossing individuals of both transinfected lines with field mosquitoes from 6 localities of Reunion Island (for which F₀ were used for crosses). Crosses involved 2-5 day-old virgin females and males (20 from each sex) in 15x15x15 cm cages (Bugdorm, Taiwan). Three replicates were performed for each crossing experiment. Females were given a blood meal 48 hours after mating and eggs were collected 5 days later. After 7 days of drying, eggs were allowed to hatch for 48 hours and the number of hatched and unhatched eggs was counted.

We used the same protocol to quantify the effect of aging on sterility: males from both transinfected lines were crossed with 2-5 dold females from the S-Run line. The effect of male aging on sterility was tested for four ages: 3-4, 7-8, 11-12, and 15-16 days. Three repetitions per age were performed. To account for embryonic mortality not related to CI, we used a corrected index of CI (CI_corr) calculated as follows: CI_corr = [(CI_obs − CCM)/(100 − CCM)] × 100, where CI_obs is the percentage of unhatched eggs observed in a given incompatible cross, and CCM is the mean mortality observed in the control crosses. The CI_corr is given for crosses involving laboratory lines (transinfected, asymbiotic, and S-Run lines), compared to crosses using F₀ from natural populations for which only incompatible crosses were performed. Thus, for these crosses, a hatch rate calculated as follows is given: Hatch rate = (number of hatched eggs / total number of eggs) x 100. The F₉ and F₄ post microinjections were used for Alb-RUN-wPip-I and Alb-RUN-wPip-IV lines, respectively.

Measure of life-history traits in transinfected lines (data file "Life history traits of the transinfected lines hatch rate and fecundity" and "Life history traits of the transinfected lines survival rate")

We standardized larvae-rearing conditions for all lines used in these experiments. Specifically, eggs from each line were allowed to hatch for 24 hours at 31°C in containers containing 250 mL of water supplemented with 50 mg TetraMin(©TETRA) and for each line 2,000 L1 were manually counted and transferred to a tray (53x325x65 cm, MORI 2A) and fed with a controlled quantity of food (tetraMin (©TETRA), day 1: 0.3 g, day 2: 0.6 g, day 3: 20 g, day 4: 25, day 5: 20 g, day 6: 15 g, day 7: 10 g) until pupal stage.

For each line, survival rate was measured by introducing 100 newly emerged males or females were placed separately in 15x15x15cm cages (Bugdorm, Taiwan). Three replicates were performed for each line and sex. The number of dead mosquitoes was recorded daily for 45 days to determine the survival rate and sugar meal (5%) was changed weekly.

For each line, the number of laid eggs was measured by placing 200 male and 200 female pupae (one cage per line) in 30x30x30 cm cage (Bugdorm, Taiwan) and left for three days after emergence. A blood meal was then provided and we randomly conserved 50 females. Five days later, females were placed individually in a small plastic cup for egg laying. Thirty cups for each line with at least one laid egg were randomly selected for the analyses. After 7 days of drying, eggs were counted, allowed to hatch for 48 hours and hatch rate was measured. 

Mating competitiveness of the Mafati line (data file "Mating competitiveness of the Mafati male")

We tested the mating competitiveness of males from the transinfected Mafati line by mixing virgin females from the S-Run line with different ratios of Mafati males. For all lines, larval rearing conditions were standardized. Eggs from both lines were allowed to hatch for 24 hours in the same conditions (at 31°C in a pot containing 250 mL of water and 50 mg of TetraMin(©TETRA)). For the S-Run line, 2,000 first instar larvae were manually counted and transferred into 53x325x65 cm trays (MORI 2A). For the Mafati line, dieldrin selection was performed on six repetitions using 1,000 manually counted L1. These L1 were exposed to dieldrin 0.08 ppm for 6 hours, rinsed with clean water, and then counted to obtain, as for the S-Run line, 2,000 L1 in a tray, which were then fed with the same quantity of food (TetraMin, ©TETRA) until pupal stage. Pupae from both lines were manually sex sorted under a binocular loupe and males and females were allowed to emerge in separate 30x30x30 cm cages (Bugdorm, Taiwan) with sugar meal (5%). Two to three day-old virgin females and males were used for this experiment. Females (N = 100) were first placed inside cages followed by the simultaneous release of all (S-Run and Mafati) males, and mating was allowed for 48 hours. All males were then removed, a blood meal was provided and eggs were collected by oviposition en masse five days later. After 7 days of drying, eggs were allowed to hatch and the hatching rate was measured and used as a proxy of mating competitiveness. Five ratios were tested: 1:1 (50♂ S-Run:50♂ Mafati), 1:5 (17♂ S-Run:85♂ Mafati), 1:10 (9♂ S-Run:90♂ Mafati) and two control ratios, 1:0 (100♂ S-Run:0♂ Mafati) and 0:1 (0♂ S-Run:100♂ Mafati). Three replicates were performed for each ratio.

Laboratory cage population suppression experiment (data file "Evaluation of Mafati males effectiveness to suppress Aedes albopictus population hatch rate" and "Evaluation of Mafati males effectiveness to suppress Aedes albopictus population number of eggs")

To establish an S-Run population in the three treatment and control cages (60x60x60 cm, Bugdorm, Taiwan), 100 pupae from each sex were introduced weekly in each cage. One week after the first introduction, three blood meals were provided weekly and eggs were collected twice a week. After 7 days of drying, all eggs were counted and allowed to hatch for 24 hours. Thus, the number of eggs and hatch rates were determined weekly for each cage. For the treatment cages, 500 male adults from the Mafati line were released weekly starting from week 4 at a 5:1 ratio (500 Mafati males: 100 S-Run males). In treatment cages, we took into account the level of population suppression induced by the repeated releases of Mafati males. Thus the number of pupae from the S-Run line introduced in the treatment cages was determined by the difference of egg hatch rate between the treatment and the control cages. For example, if the egg hatch rate in week 8 was 80% and 60% (20% difference) in control and treatment cages respectively, we introduced 20% fewer S-Run males and females in the treatment cages the following week (100 x 0.2=80), therefore 80 males and females pupae, instead of 100 in the control cages.

For this experiment, Mafati males were produced as follows. Eggs were brushed and weighed to obtain 135 mg of eggs that were placed in a 200 mL plastic container containing 30 mL of hatching solution (tap water at 5% of TetraMin (©TETRA)) and left for 8 hours for hatching. First instar larvae were then exposed for 16 hours to 0.1 ppm of dieldrin, then rinsed with clean water and transferred to a tray (2,000 L1/tray) and fed with the same quantity of food as the other lines (TetraMin, ©TETRA) until pupal stage. This protocol eliminated ~95% of females. Pupae were collected and sex separated using a pupae sex sorter (Wolbaki, WBK-P0001-V1 model) allowing us to remove the remaining females. All male pupae were then counted using the mosquito larvae/pupae counter (Radiation General Ltd, Budapest, Hungary) and used for the experiment.