Abstract
BackgroundThe leaves of carnivorous pitcher plants harbor diverse communities of inquiline species, including bacteria and larvae of the pitcher plant mosquito (Wyeomyia smithii), which aid the plant by processing captured prey. Despite the growing appreciation for this microecosystem as a tractable model in which to study food web dynamics and the moniker of W. smithii as a ‘keystone predator’, very little is known about microbiota acquisition and assembly in W. smithii mosquitoes or the impacts of W. smithii-microbiota interactions on mosquito and/or plant fitness.ResultsIn this study, we used high throughput sequencing of bacterial 16S rRNA gene amplicons to characterize and compare microbiota diversity in field- and laboratory-derived W. smithii larvae. We then conducted controlled experiments in the laboratory to better understand the factors shaping microbiota acquisition and persistence across the W. smithii life cycle. Methods were also developed to produce axenic (microbiota-free) W. smithii larvae that can be selectively recolonized with one or more known bacterial species in order to study microbiota function. Our results support a dominant role for the pitcher environment in shaping microbiota diversity in W. smithii larvae, while also indicating that pitcher-associated microbiota can persist in and be dispersed by adult W. smithii mosquitoes. We also demonstrate the successful generation of axenic W. smithii larvae and report variable fitness outcomes in gnotobiotic larvae monocolonized by individual bacterial isolates derived from naturally occurring pitchers in the field.ConclusionsThis study provides the first information on microbiota acquisition and assembly in W. smithii mosquitoes. This study also provides the first evidence for successful microbiota manipulation in this species. Altogether, our results highlight the value of such methods for studying host-microbiota interactions and lay the foundation for future studies to understand how W. smithii-microbiota interactions shape the structure and stability of this important model ecosystem.
Highlights
The leaves of carnivorous pitcher plants harbor diverse communities of inquiline species, including bacteria and larvae of the pitcher plant mosquito (Wyeomyia smithii), which aid the plant by processing captured prey
Our results show that axenic W. smithii larvae fed sterilized diet under a standard photoperiod and sterile conditions fail to grow beyond the first instar but develop normally when inoculated with the mixed community of bacteria present under conventional rearing conditions
That (i) W. smithii first instars hatched from surface-sterilized eggs contain no bacteria or fungi, as demonstrated by our inability to generate viable cultures from pooled homogenates of axenic larvae or PCR amplicons using DNA template from axenic larvae and universal bacterial and fungal primers, (ii) almost all of the ASVs we identified in our surface-sterilized larval samples were present in the water from which larvae were collected from, and (iii) several of the abundant community members we identified via high-throughput sequencing were able to individually colonize the larval gut strongly suggests that most of the bacteria we identified in whole-body W. smithii larvae and adults via sequencing were present in the digestive tract
Summary
The leaves of carnivorous pitcher plants harbor diverse communities of inquiline species, including bacteria and larvae of the pitcher plant mosquito (Wyeomyia smithii), which aid the plant by processing captured prey. While recent studies have used surveys at geographical scales to parameterize the contribution of W. smithii mosquitoes to ecosystem scale processes [12, 13], the species is rarely maintained in continuous laboratory culture. This has led to a lack of studies seeking to experimentally validate and functionally characterize W. smithii-mediated effects in the S. purpurea system
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