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. 2018 Oct 26;9(1):4464.
doi: 10.1038/s41467-018-07014-2.

Generation of axenic Aedes aegypti demonstrate live bacteria are not required for mosquito development

Maria A Correa  1 , Brian Matusovsky  2 , Doug E Brackney  3 , Blaire Steven  4
Affiliations

Generation of axenic Aedes aegypti demonstrate live bacteria are not required for mosquito development

Maria A Correa et al. Nat Commun. .

Abstract

The mosquito gut microbiome plays an important role in mosquito development and fitness, providing a promising avenue for novel mosquito control strategies. Here we present a method for rearing axenic (bacteria free) Aedes aegypti mosquitoes, consisting of feeding sterilized larvae on agar plugs containing a high concentration of liver and yeast extract. This approach allows for the complete development to adulthood while maintaining sterility; however, axenic mosquito's exhibit delayed development time and stunted growth in comparison to their bacterially colonized cohorts. These data challenge the notion that live microorganisms are required for mosquito development, and suggest that the microbiota's main role is nutritional. Furthermore, we colonize axenic mosquitoes with simplified microbial communities ranging from a single bacterial species to a three-member community, demonstrating the ability to control the composition of the microbiota. This axenic system will allow the systematic manipulation of the mosquito microbiome for a deeper understanding of microbiota-host interactions.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Experimental setup. Individual larvae were reared in the wells of a six-well plate. This picture was taken 5 days post hatching. The sterile control group consisted of autoclaved liver:yeast extract not embedded in an agar plug, which does not support larval development (individual larvae in this group are too small to see at this scale). Any development of sterile control larvae was taken as a sign of contamination and the experiment was discarded. Because of the delay in development, the axenic larvae are smaller than the gnotobiotic or CM groups. The scale bar at the top of the figure denotes 3.5 cm. The red arrow and text indicate the LY plug. The red circles highlight the individual larvae in the wells
Fig. 2
Fig. 2
PCR detection of bacterial DNA. Total DNA was extracted from axenic (A), axenic supplemented with heat-killed E. coli (AS), and colony microbiome (CM) mosquitoes at three different developmental stages (larva, non-blood fed adult, and blood fed adult). DNA from three individuals from each group was used as a template for amplification of bacterial 16S rRNA genes. A and AS mosquitoes show no visible PCR products across all developmental stages, whereas amplification products were identified in the CM mosquitoes. A positive control (+) containing amplified E. coli K-12 DNA and a non-template control (NTC) are also included on the gel. Amplification consisted of 30 cycles and an annealing temperature of 55 °C (see Methods)
Fig. 3
Fig. 3
Delayed development in axenic larvae. Time to pupation is shown for axenic (A), axenic supplemented with heat-killed E. coli (AS), gnotobiotic (G), and colony microbiome (CM) larvae. Points represent time to pupation for individual larvae. The experiment was performed with 18 individuals per treatment (three six-well plates), with three independent replications (i.e., 54 individuals). The central red line denotes the mean pupation time, with the bars indicating the standard error of the mean. Statistically significant differences between groups were identified with the Kruskall–Wallis test and columns labeled with different letters were significantly different with a P-value < 0.05
Fig. 4
Fig. 4
Biometric assessment of adult mosquitoes. a Male:female ratio for axenic (A), axenic supplemented with heat-killed E. coli (AS), gnotobiotic (G), and colony microbiome (CM) mosquitoes. Bars marked with a different letters were significantly different in sex ratio with a P-value < 0.05 by Fisher’s Exact test. The number of mosquitoes assayed is indicated above the bars. b Female wing length. c Male wing length. Points represent wing lengths of individual mosquitoes (n = 54). Mean wing length and standard error of all individuals are signified by the red bars. The numbers of mosquitoes assayed are the same as indicated in panel A. Columns labeled with different letters were significantly different with a P-value of < 0.05 as determined by a one-way analysis of variance
Fig. 5
Fig. 5
Mosquito survival curves. a Adult survival as measured days post emergence. b Mosquito survival curve corrected for larval development time. For both a and b, analysis of the Kaplan–Meier survival curves by the Logrank test revealed statistical significance when all groups were compared together (P < 0.0001). Sample sizes varied between groups; AS (n = 23), A (n = 21), G (n = 17), and CM (n = 27). All pairwise comparisons were also significantly different when compared using the Logrank Mantel–Cox test (P < 0.0001)
Fig. 6
Fig. 6
Egg clutch size. The number of eggs per mosquito is shown for axenic (A; n = 12), axenic supplemented with heat-killed E. coli (AS; n = 15), gnotobiotic (G; n = 12), and colony microbiome (CM; n = 14) mosquitoes. Points represent clutch size for individual females. Mean clutch size and standard error of all individuals are signified by the red bars. No significant differences between the groups was detected using a one-way analysis of variance with a P-value cutoff of <0.05
Fig. 7
Fig. 7
Strain abundance in mosquitoes colonized by simplified microbial communities. Each point indicates the colony forming units (cfu) of each of three strains that were introduced to individual a axenic larvae or b axenic adults. The red horizontal bar represents the mean with error bars indicating the standard error of the mean. Counts were determined from 6 individuals for both the larvae and adults. Columns labeled with different letters were significantly different as determined by a one-way analysis of variance with a P-value cutoff of <0.05
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