The microbiota impacts life history traits and mating success in male Aedes aegypti mosquitoes

El-Dougdoug, Noha1 ; Magistrado, Dom1 ; Perry, Kayla1 ; Short, Sarah 1

Published Sep 03, 2026 on Dryad. https://doi.org/10.5061/dryad.bk3j9kds4

Data files

Sep 03, 2026 version files 452.06 KB

FileS1_AXMXLC_AllRawData.xlsx

113.57 KB
fileS2_Rcode_Routput.pdf

327.05 KB
README.md

11.44 KB

Abstract

Aedes aegypti mosquitoes transmit multiple arboviruses, but population suppression through the mass release of sterile or incompatible male mosquitoes can effectively reduce populations. These methods depend on the reliable mass-rearing of healthy, robust males that can successfully mate with wild females. The microbiota, a critical component of the larval diet, can dramatically influence life history traits relevant to mass-rearing and male quality. Here, we used axenic (microbe-free), monoxenic (inoculated with E. coli), and “laboratory community” mosquitoes (inoculated with an undefined microbiota derived from laboratory mosquitoes) to show that longevity was significantly enhanced in axenic and monoxenic males compared to laboratory community males. Moreover, monoxenic males more efficiently obtained mates in non-competitive mating scenarios compared to laboratory community males. However, microbiota treatment had no effect when males from different treatments competed for a mate. Our findings suggest that the microbiota is a key determinant of male mosquito life history with direct implications for optimizing production of males for control programs.

Dataset DOI: 10.5061/dryad.bk3j9kds4

Description of the data and file structure

We designed three experiments to investigate the impact of microbiota modulation or microbiota absence on life history traits of unsexed larvae and adult male Ae. aegypti. In all experiments, we used Thailand (Thai) strain Ae. aegypti mosquitoes. After hatching larvae from sterilized eggs, the axenic larvae were randomly allocated to one of three treatments according to the objectives of each experiment: axenic (AX), i.e. maintained in a microbiota-free state throughout the experiment; monoxenic (MX), i.e. inoculated with only E. coli; or lab community (LC), i.e. mosquitoes inoculated with an undefined microbiota derived from laboratory reared mosquitoes.

We then collected data on pupation time, eclosion time, wing length, adult longevity, starvation resistance, and mating success under non-competitive and competitive scenarios.

Files and variables

File: FileS1_AXMXLC_AllRawData.xlsx

Description: All raw data collected for the project in the form of csv files combined into a single excel workbook.

Sheet 1 "Fig3A_MX_LC_Pupation" Variables:

treatment: bacterial treatment (MX, or LC)
microbial community: type of bacteria added to the larval water (lw = larval water, s17=E. coli S17)
flask: each experiment was replicated over multiple flasks of larvae; this variable indicates which flask the larvae was derived from.
individual: individual larval number within flask
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
daysposthatching: day post hatching at which pupation occurred.
status: marks an event (event = 1, i.e. pupation)

Sheet 2 "Fig3B_MX_LC_Eclosion" Variables:

treatment: bacterial treatment (MX, or LC)
microbial community: type of bacteria added to the larval water (lw = larval water, s17=E. coli S17)
flask: each experiment was replicated over multiple flasks of larvae, this variable indicates which flask the larvae was derived from. (NA = not applicable)
individual: individual number within flask
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
daysposthatching: day post hatching at which eclosion occurred.
dayspostpupation: day post pupation at which eclosion occurred.
status: marks an event (event = 1, i.e. eclosion)

Sheet 3 "Fig3C_MX_LC_Survival_SugarFed" Variables:

treatment: bacterial treatment (MX, or LC)
microbial community: type of bacteria added to the larval water (lw = larval water, s17=E. coli S17)
individual: individual number of males under survival monitoring
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
daysposteclosion: day post eclosion at which death occurred.
status: marks an event (event = 1, i.e. death)

Sheet 4 "Fig3D_MX_LC_Survival_WaterFed" Variables:

treatment: bacterial treatment (MX, or LC)
microbial community: type of bacteria added to the larval water (lw = larval water, s17=E. coli S17)
individual: individual number of males under survival monitoring
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
daysposteclosion: day post eclosion at which death occurred.
status: marks an event (event = 1, e.g. death)

Sheet 5 " Fig4_S678_MX_LC_Wings_Cop_Inse" Variables:

id: individual adult number within treatment.
treatment: bacterial treatment (MX, or LC)
malewinglength: length of male wings in mm. (NA = not applicable)
femwinglength: length of female wings in mm. (NA = not applicable)
copulaformation: whether or not copulation occurred (1) or did not occur (0)
spermtransfer: whether or not sperm transfer occurred (1) or did not occur (0). (NA = not applicable)
timetocopula: time in s from start of mating trial until mating occurred. (NA = not applicable)
replicate_original: original numbering of full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
replicate: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).

Sheet 6 "Fig5_MX_LC_ GrpMtng_Binomial" Variables:

id: individual adult number within treatment.
treatment: bacterial treatment (MX, or LC)
minutes: number of minutes mating trial was allowed to span.
replicate: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
spermtransfer: whether or not sperm transfer occurred (1) or did not occur (0).
date: date of experiment (day-month-year)
trtrep: concatenated treatment/replicate designation

Sheet 7 "Fig6_MX_LC_MatingCompetition" Variables:

rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
trial: number of mating events conducted for each male individual (i.e. each trial represents one experimental opportunity for a male to mate with a female).
lc_win: whether or not LC males won (1) or lost (0) the mating trial
mx_win: whether or not MX males won (1) or lost (0) the mating trial

Sheet 8 "Fig7_MX_LC_BacterialLoad_0day" Variables:

treatment: bacterial treatment (MX, or LC)
days: days post addition of bacteria to larval flasks
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
cfuperml: Colony forming units per mL
logcfuperml: log10 of cfuperml

Sheet 9 " Fig7_S9_S10_MX_LC_BacterialLoad" Variables:

treatment: bacterial/larval treatment (LC+larvae, LC, MX+larvae, MX)
bacttreat: bacterial treatment (MX, or LC)
larvae: whether larvae are present or absent in the flask
days: days post addition of bacteria to larval flasks
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
cfuperml: Colony forming units per mL
logcfuperml: log10 of cfuperml
flask: each experiment was replicated over multiple flasks of larvae, this variable indicates which flask the larvae/measurement was derived from.
flaskno: number of the flask that indicates which flask the larvae/measurement was derived from.

Sheet 10 " Fig8A_AX_MX_LC_Pupation" Variables:

treatment: bacterial treatment (AX, MX, or LC)
plate: plate in which larvae were reared (when reared in 6-well plates)
individual: individual larval number within plate
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
daysposthatching: day post hatching at which pupation occurred.
status: marks an event (event = 1, i.e. pupation)
notes: note for status (0) (i.e. 0 indicates larvae did not pupate within the experimental timeline)

Sheet 11 " Fig8B_AX_MX_LC_Eclosion" Variables:

treatment: bacterial treatment (AX, MX, or LC)
plate: plate in which larvae were reared (when reared in 6-well plates)
individual: individual larval number within plate
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
daysposthatching: day post hatching at which eclosion occurred.
status: marks an event (event = 1, i.e. eclosion).

Sheet 12 " Fig8C_AX_MX_LC_Survival" Variables:

treatment: bacterial treatment (AX, MX, or LC)
microbial community: type of bacteria added to the larval water (neg=nothing, lw = larval water, s17=E. coli S17)
individual: individual number of adult males under survival monitoring.
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
daysposteclosion: day post eclosion at which death occurred.
status: marks an event (event = 1, i.e. death)

Sheet 13 " Fig8D_AX_MX_LC_WingLength" Variables:

id: individual adult male number within treatment.
treatment: bacterial treatment (AX, MX, or LC)
malewinglength: length of male wings in mm
replicate: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).

Sheet 14 " Fig9_MX_Ampicillin_Survival" Variables:

treatment: ampicillin/bacterial treatment (amp200ecoli, amp200, ampnegecoli, ampneg)
amp: ampicillin treatment (200ug = treated with 200ug ampicillin, and neg=untreated with ampicillin)

e.coli: type of bacteria that infected the adult male (neg=noninfected, and pos=infected)

individual: individual number of adult males under survival monitoring
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
dayspostinfection: day post infection at which death occurred.
status: marks an event (event = 1, i.e. death)

Sheet 15 " FigS11_MX_Ampicillin_Pupation" Variables:

treatment: ampicillin treatment (amptrt, untreat)

amp: ampicillin treatment (200ug = treated with 200ug ampicillin, and neg=untreated with ampicillin)

source: flask that larvae was derived from; each experiment was replicated over multiple flasks of larvae.
individual: individual larval number within flask
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
daysposthatching: day post hatching at which pupation occurred.
status: marks an event (event = 1, i.e. pupation)

Sheet 16 " FigS12_MX_Ampicillin_Eclosion" Variables:

treatment: ampicillin treatment (amptrt, untreat)
amp: ampicillin treatment (200ug = treated with 200ug ampicillin and neg=untreated with ampicillin).
source: flask that larvae was derived from; each experiment was replicated over multiple flasks of larvae.
individual: individual larval number within flask
rep: full biological replicate of the experiment (i.e. each time the experiment was replicated in full, starting from hatching of new mosquito eggs).
daysposthatching: day post hatching at which eclosion occurred.
status: marks an event (event = 1, i.e. eclosion)

File: fileS2_Rcode_Routput.pdf

Description: All code used to conduct statistical analyses and create figures.

Code/software

Analyses were performed in R and R studio using packages survival, survminer, car, ggplot2, interactions, arm, stats, dplyr, tidyverse, ggpubr, rstatix, lavaan, and DHARMa.

All code used to analyze data and generate figures can be found in "fileS2_Rcode_Routput.pdf"

Access information

Other publicly accessible locations of the data:

Data was derived from the following sources: