DOI: 10.5061/dryad.s7h44j1f8
title: Combining transinfected Wolbachia and a genetic sexing strain to control Aedes albopictus in laboratory-controlled conditions

description: 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.
description: <strong>Crossing experiments and effect of male age on CI
 intensity (data file "<em>CI expression of Wolbachia
 transinfected lines in Aedes albopictus</em>" and
 "<em>Effect of male age on CI
 intensity</em>"</strong> To evaluate
 CI penetrance in both transinfected lines, we performed reciprocal
 <em>en masse</em> 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 F<sub>0</sub> 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<sub>corr</sub>) calculated as
 follows: CI<sub>corr</sub> = [(CI<sub>obs</sub> −
 CCM)/(100 − CCM)] × 100, where CI<sub>obs</sub> 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<sub>corr</sub> is given for crosses involving laboratory
 lines (transinfected, asymbiotic, and S-Run lines), compared to crosses
 using F<sub>0</sub> 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 F<sub>9</sub> and
 F<sub>4</sub> post microinjections were used for
 Alb-RUN-<em>w</em>Pip-I and
 Alb-RUN-<em>w</em>Pip-IV lines, respectively.
 <strong>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")</strong>
 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.<strong> </strong>
 <strong>Mating competitiveness of the Mafati line (data
 file "Mating competitiveness of the Mafati
 male")</strong> 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 <em>en
 masse</em> 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.
 <strong>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")</strong> 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.
description: # Combining transinfected Wolbachia and a genetic sexing strain to control
 *Aedes albopictus* in laboratory-controlled conditions ## Datasets
 included: 1\) CI expression of Wolbachia transinfected lines in *Aedes
 albopictus* * Different crosses were performed to quantify the cytoplasmic
 incompatibility (CI): variable "crosses (malexfemale)" * The
 number of hatched and unhatched eggs and the hatch rate (%) observed in
 these crosses: variable "nb of eggs", "nb of hatched
 eggs", "nb of unhatched eggs", "hatch rate (%)" *
 The percentage of CI and the corrected CI observed in these crosses:
 variable "CI (%)", "CIcorr (%)" * When the CIcorr was
 not calculated, "NA" was indicated 2\) Effect of male age on CI
 intensity * Different crosses were performed to quantify the male age
 effect on cytoplasmic incompatibility (CI): variable "crosses" *
 The different ages of males tested: variable "age of male" * The
 number of hatched, unhatched eggs and the hatch rate (%) observed in these
 crosses: variable "nb of eggs", "nb of hatched eggs",
 "nb of unhatched eggs", "hatch rate (%)" * The
 percentage of cytoplasmic incompatibility (CI) and the corrected CI
 observed in these crosses: variable "CI (%)", "CIcorr
 (%)" * The CIcorr cannot be calculated for control crosses, thus
 "NA" was indicated 3\) Life history traits of the transinfected
 lines hatch rate and fecundity * Different lines of mosquitoes used to
 measure the fecundity (number of eggs laid): variable "lines" *
 The number of hatched and unhatched eggs and the hatch rate (%) observed
 for each line: variable "nb of eggs", "nb of hatched
 eggs", "nb of unhatched eggs", "hatch rate (%)"
 4\) Life history traits of the transinfected line survival rate * The
 number of days for which mortality was recorded: variable "time"
 * Different lines of mosquitoes used to measure the survival rate * The
 number of mosquitoes still alive when mortality was recorded: variable
 "nb of survivor" * The number of mosquitoes dead when mortality
 was recorded: variable "nb of death" * The sex (male or female)
 used for each line of mosquitoes * A variable "status" was added
 to indicate when the mortality recording was ongoing (value 1) or closed
 (value 0) 5\) Mating competitiveness of the Mafati male * The different
 ratios of incompatible males used to evaluate the sexual competitiveness
 of Mafati males: variable "ratio" * The number of hatched,
 unhatched eggs and the hatch rate (%) observed for each ratio tested:
 variable "nb of eggs", "nb of hatched eggs", "nb
 of unhatched eggs", "hatch rate (%)" 6\) Evaluation of
 Mafati male's effectiveness in suppressing the *Aedes albopictus*
 population * The number of weeks during which the number of eggs laid and
 the hatch rate of eggs were measured: variable "week" * The two
 different conditions tested: control (without incompatible males) and
 treatment (with incompatible males). Variable: "condition" * The
 number of eggs collected each week: variable "nb of eggs" * The
 hatch rate of eggs collected each week: variable "hatch rate(%)"