Dissection of Mosquito Ovaries, Midgut, and Salivary Glands for Microbiome Analyses 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.

Our research focuses on microbiome community in mosquito at the scale of individual organs. Using metagenomic analysis, we study symbionts in ovaries, midgut and salivary glands. By preventing cross-contamination during dissections, we aim to study how these microbes affect mosquito biology at front scale, and adding targeted control methods.

Working with individual organs is really complex because we need to work on them intact without causing any damage or contamination between organs. Plus, we only have a small amount of genetic material available. Research on vector mosquitoes typically focuses on the wall organism.

However, interactions between viruses, bacteria, and mosquitoes can differ depending on the specific organ. By carefully dissecting and isolating mosquito organs, we can study each compartment individually and gain a better understanding of how these interactions function. To begin, obtain surface sterilized mosquitoes in a 1.5 milliliter tube containing PBS.

Transfer a cleaned mosquito onto a drop of sterile 1X PBS placed on the microscope slide. Using tweezers, hold the mosquito's thorax to prevent it from moving. Then place a needle at the neck just below the head and gently pull on the mosquito's head to separate it from the thorax.

Using a needle, cut the salivary glands at the level of the salivary duct. Retrieve the glands with the needle and place them into a new drop of PBS to rinse. Next, employing a needle, retrieve the salivary glands.

Place the mosquito on its back to begin dissecting the ovaries. Using forceps, hold the mosquito by the thorax to prevent movement and puncture the third abdominal segment, starting from the bottom, with a needle. While piercing the third abdominal segment, pull down the abdomen to open it and reveal the internal organs.

Remove any remaining pieces of the mosquito exoskeleton to facilitate organ release. Retrieve both ovaries with sterilized forceps and place them into a new drop of sterile PBS on the microscope slide to rinse. Transfer the rinsed ovaries into a 1.5 milliliter tube containing 100 microliters of preservation buffer.

Next, using a sterile needle, separate the midgut from the foregut and hindgut. Remove the Malpighian tubules from the midgut and place the midgut into a new drop of sterile PBS to rinse it and prevent contamination. Then transfer the midgut to another drop of sterile PBS.

With forceps open the midgut to release bacteria. Pipette all the PBS-containing bacteria and the midgut tissue into a new sterilized 1.5 milliliter tube containing 100 microliters of preservation buffer. Add five microliters of PBS onto the same area of the microscope slide where the midgut was previously placed.

Pipette the PBS back into the same 1.5 milliliter tube to retrieve any bacteria that may have stuck to the slide. Finally, use sterile forceps to grasp the mosquito carcass and store it in a sterilized 1.5 milliliter tube containing 100 microliters of preservation buffer.