Manipulating the Mosquito Microbiota to Study Its Function

Romoli, Ottavia; Gendrin, Mathilde

doi:10.1007/978-1-0716-0259-1_11

Cited by 5 publications

(7 citation statements)

References 18 publications

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“…aegypti larvae, and that S. marcescens also secretes proteases and chitinases which have a larvicidal activity ( Jupatanakul et al, 2020 ). On the other side, bacteria are required for normal larval development, and we have set up methods to test whether specific bacteria are able to support larval development ( Coon et al, 2014 ; Romoli and Gendrin, 2020 ). Using our protocol, 100% sterile larvae are blocked at the first instar unless bacteria are added in the medium.…”

Section: Resultsmentioning

confidence: 99%

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The Effect of Secondary Metabolites Produced by Serratia marcescens on Aedes aegypti and Its Microbiota

et al. 2021

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Serratia marcescens is a bacterial species widely found in the environment, which very efficiently colonizes mosquitoes. In this study, we isolated a red-pigmented S. marcescens strain from our mosquito colony (called S. marcescens VA). This red pigmentation is caused by the production of prodigiosin, a molecule with antibacterial properties. To investigate the role of prodigiosin on mosquito-S. marcescens interactions, we produced two white mutants of S. marcescens VA by random mutagenesis. Whole genome sequencing and chemical analyses suggest that one mutant has a nonsense mutation in the gene encoding prodigiosin synthase, while the other one is deficient in the production of several types of secondary metabolites including prodigiosin and serratamolide. We used our mutants to investigate how S. marcescens secondary metabolites affect the mosquito and its microbiota. Our in vitro tests indicated that S. marcescens VA inhibits the growth of several mosquito microbiota isolates using a combination of prodigiosin and other secondary metabolites, corroborating published data. This strain requires secondary metabolites other than prodigiosin for its proteolytic and hemolytic activities. In the mosquito, we observed that S. marcescens VA is highly virulent to larvae in a prodigiosin-dependent manner, while its virulence on adults is lower and largely depends on other metabolites.

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Section: Resultsmentioning

confidence: 99%

“…Larval development assays were performed as described previously ( Romoli and Gendrin, 2020 ). Eggs were sterilized to obtain axenic larvae.…”

Section: Methodsmentioning

confidence: 99%

The Effect of Secondary Metabolites Produced by Serratia marcescens on Aedes aegypti and Its Microbiota

et al. 2021

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“…Depending on the experiment type, two rearing methods were used: for experiments aiming to analyse adult mosquitoes, a batch rearing in cell-culture flasks (10-15 larvae per flask) was used; for experiments requiring the follow-up of single larvae, larvae were placed in single wells of sterile 24-well plates 49 .…”

Section: Generation Of Gnotobiotic Larvae and Axenic Adults Via Revermentioning

confidence: 99%

“…Imaging was performed on an Euromex dissection microscope equipped with a camera. For each larva, the distance between the anterior border of the head and the posterior border of the last abdominal segment excluding the syphon 49 was measured using the ImageFocus software (version 4, Euromex). Four independent mosquito batches were examined and, for each replicate, at least 27 individuals per condition were analysed.…”

Section: Generation Of Gnotobiotic Larvae and Axenic Adults Via Revermentioning

confidence: 99%

“…Wings from several individuals were dissected and placed on a microscope slide. The distance between the alular notch and the radius 3 vein was measured for each wing using a dissection microscope equipped with a camera 49 . Four independent mosquito batches were examined and, for each replicate, 7-20 individuals per condition were analysed.…”

Section: Generation Of Gnotobiotic Larvae and Axenic Adults Via Revermentioning

confidence: 99%

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Production of germ-free mosquitoes via transient colonisation allows stage-specific investigation of host–microbiota interactions

et al. 2021

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The mosquito microbiota impacts the physiology of its host and is essential for normal larval development, thereby influencing transmission of vector-borne pathogens. Germ-free mosquitoes generated with current methods show larval stunting and developmental deficits. Therefore, functional studies of the mosquito microbiota have so far mostly been limited to antibiotic treatments of emerging adults. In this study, we introduce a method to produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with bacteria genetically modified to allow complete decolonisation at any developmental stage. We show that, unlike germ-free mosquitoes previously produced using sterile diets, reversibly colonised mosquitoes show no developmental retardation and reach the same size as control adults. This allows us to uncouple the study of the microbiota in larvae and adults. In adults, we detect no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, data from our transcriptome analysis and diet supplementation experiments following decolonisation suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development.

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Production of germ-free mosquitoes via transient colonisation allows stage-specific investigation of host-microbiota interactions

Romoli

Schönbeck

Hapfelmeier

et al. 2019

Preprint

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15The mosquito microbiota impacts the physiology of its host and is essential for normal larval 16 development, thereby influencing transmission of vector-borne pathogens. Germ-free 17 mosquitoes generated with current methods show larval stunting and developmental deficits. 18Therefore, functional studies of the mosquito microbiota have so far mostly been limited to 19 antibiotic treatments of emerging adults. In this study, we developed a novel approach to 20 produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with 21 bacteria genetically modified to allow complete decolonisation at the end of larval 22Overall, this study provides a new tool to study the microbiota in insects and deepens our 29 knowledge on the metabolic contribution of bacteria to mosquito development. 30We developed a transient bacterial colonisation method to support larval development while 56 producing fully developed germ-free adults. Using this approach, we were able to support 57 development within a standard timeline and without affecting adult size, showing that these 58 germ-free individuals represent a valid tool for mosquito microbiota studies. We further used 59 transient colonisation during early larval development to characterise the role of bacteria in 60 larval development using a transcriptomic approach and diet complementation. Our data 61 indicate that bacteria contribute to folate biosynthesis and energy storage and that folic acid 62 supports larval development in the absence of a microbiota. 63 64 Results 65 66 Production of germ-free adult mosquitoes via transient bacterial colonisation 67After microbiological sterilisation of mosquito eggs, hatchlings provided with autoclaved 68 standard rearing diet are able to survive for weeks but remain first-instar larvae. Their 69 development can be rescued by bacterial colonisation, for instance with Escherichia coli 4,10 . 70 Therefore, we hypothesised that transient bacterial colonisation would be an efficient way to 71 support larval development and produce germ-free adults. To test this, we used HA416, an E. 72 coli HS-derived strain that is auxotrophic for two non-standard amino-acids required for 73 peptidoglycan synthesis (meso-diaminopimelic acid, m-DAP, and D-alanine, D-Ala) and that 74 was previously used to transiently colonise the gut of germ-free mice 12 . We supplemented the 75 food of gnotobiotic larvae with m-DAP and D-Ala to support the proliferation of auxotrophic 76 E. coli during larval development only ( Figure 1A). These supplements were toxic to first instar 77 larvae when provided at the concentrations previously used in vitro, but 4-fold lower 78 concentrations supported growth of auxotrophic E. coli (Supplementary Figure S1) without 79 causing toxicity in larvae. The auxotrophic E. coli strain rescued the development of axenic 80

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Manipulating the Mosquito Microbiota to Study Its Function

Cited by 5 publications

References 18 publications

The Effect of Secondary Metabolites Produced by Serratia marcescens on Aedes aegypti and Its Microbiota

The Effect of Secondary Metabolites Produced by Serratia marcescens on Aedes aegypti and Its Microbiota

Production of germ-free mosquitoes via transient colonisation allows stage-specific investigation of host–microbiota interactions

Production of germ-free mosquitoes via transient colonisation allows stage-specific investigation of host-microbiota interactions

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