Name: Video: Organ-Specific Dissection of Mosquitoes for Microbiota and Metagenomic Analysis
Uploaded: 2024-10-04T14:25:00
Duration: 2 min 48 s
Description: 309 Views. Organ-Specific Dissection of Mosquitoes for Microbiota and Metagenomic Analysis

organ-specific dissection of mosquitoes for microbiota and metagenomic analysis

High-Precision Mosquito Organ Isolation for Microbiome Analyses

Mosquitoes spread a wide range of pathogens causing diseases and are a serious threat to public health. Due to an increased prevalence of insecticide resistance among mosquito populations and in the absence of effective vaccines against these pathogens, new biocontrol methods that focus on the mosquito microbiome are emerging. In particular, the intracellular bacterium Wolbachia, which can interfere with pathogen transmission and manipulate host reproduction, stands out1,2,3. In addition, other mosquito symbionts are central to the survival, development, or immune system of their host, as well as in infection and transmission of pathogens, and show great promise for their exploitation to fight vector-borne diseases4,5,6,7,8.
Microorganisms associated with mosquitoes span all domains of microbial life (including bacteria, eukaryotes, and fungi) that interact intimately with their host but also with each other in the different body compartments9,10. Therefore, a better understanding of the microbiota, its potential seasonal variation11, and the mechanisms by which its members interact directly in distinct mosquito tissues can help develop new targeted biocontrol methods or improve existing ones. However, the majority of symbionts remain resistant to cultivation, rendering their characterization impossible.
The advent of second- and third-generation sequencing methods, together with state-of-the-art assembly and binning approaches, have enabled the reconstruction of microbial genomes and accessing the diversity and functional potential of non-cultivable microbes. Here, we present a method for the dissection of the ovaries, midgut, and salivary glands of female mosquitoes while preventing cross-contamination. This protocol can be followed by genomic DNA extraction and subsequent metabarcoding or shotgun metagenomic sequencing to explore the diversity and function of mosquito microbiota at the organ level. We provide an example of mosquito dissection and microbiome data for Culex spp. specimens although this protocol can be extended to vectors of other genera&#160;such as Anopheles or Aedes.

Author Spotlight: Optimizing Mosquito Organ Dissection for Studying Symbionts and Vector Microbiomes

Dissection of Mosquito Ovaries, Midgut, and Salivary Glands for Microbiome Analyses at the Organ Level

Organ-Specific Dissection of Mosquitoes for Microbiota and Metagenomic Analysis

organ-specific-dissection-mosquitoes-for-microbiota-metagenomic

Research

JoVE Journal

Biology

309 Views. Organ-Specific Dissection of Mosquitoes for Microbiota and Metagenomic Analysis

Video: Organ-Specific Dissection of Mosquitoes for Microbiota and Metagenomic Analysis

The global burden of mosquito-transmitted diseases, including malaria, dengue, West Nile, Zika, Usutu, and yellow fever, continues to increase, posing a significant public health threat. With the rise of insecticide resistance and the absence of effective vaccines, new strategies are emerging that focus on the mosquito's microbiota. Nevertheless, the majority of symbionts remain resistant to cultivation. Characterizing the diversity and function of bacterial genomes in mosquito specimens, therefore, relies on metagenomics and subsequent assembly and binning strategies. The obtention and analysis of Metagenome-Assembled Genomes (MAGs) from separated organs can notably provide key information about the specific role of mosquito-associated microbes in the ovaries (the reproductive organs), the midgut (key for food digestion and immunity), or the salivary glands (essential for the transmission of vector-borne diseases as pathogens must colonize them to enter the saliva and reach the bloodstream during a blood meal). These newly reconstructed genomes can then pave the way for the development of novel vector biocontrol strategies. To this aim, it is required to isolate mosquito organs while avoiding cross-contamination between them or with microorganisms present in other mosquito organs. Here, we describe an optimized and contamination-free dissection protocol for studying mosquito microbiome at the organ level.

Microbial communities in mosquitoes hold great promise for vector biocontrol strategies. Most symbionts are uncultivable, requiring metagenomic analyses. We describe a method to dissect female mosquitoes and separate ovaries, midgut, and salivary glands preventing cross-contamination, facilitating microbiome studies at the organ level, and enhancing understanding of microorganisms' roles in mosquito biology.

The global burden of mosquito-transmitted diseases, including malaria, dengue, West Nile, Zika, Usutu, and yellow fever, continues to increase, posing a ...