The overall goal of this experiment is to extract endosymbionts from tiny insects for further experiments. This method can help answer key questions in the entomology and the macrobiology view because most endosymbionts cannot be cultured in vitro. Method show isolate adequate quantities of bacteria for further experiments are very important.
The main advantage of this technique is that we can conveniently isolate whitefly bacterial endosymbionts. This can also be applied to the isolation of endosymbionts from other insects such as aphids, plant hoppers, and thrips. To begin, collect an individual adult whitefly and homogenize it in 30 microliters of lysis buffer.
Incubate the homogenate at 65 degrees Celsius for one hour. Then 100 degrees Celsius for 10 minutes. Using whitefly DNA, and mitochondial cytochrome oxidase 1 primers, prepare 25 microliter PCR reactions using the following reagents.
Then, run the PCR reactions using the program shown here. Next, use a DNA gel extraction kit following the recommended protocol to clean the PCR product. Then, perform sequence analysis according to the text protocol.
To amplify specific genes of each endosymbiont within the whitefly, carry out PCR on whitefly DNA. According to a previously published protocol, subject the amplified sample to agarose gel electrophoresis and sequencing to determine the bacterial species within whitefly and identify the specific gene of each bacterium. To dissect and purify the whitefly bacteriome, add 100 microliters of 1x PBS solution onto a microscope slide.
Then, pick third or fourth instar nymphs from cotton leaves. And immerse them into the PBS. Under a microscope, use fine entomological needles to pull the bacteriomes out from the whitefly body.
Use care when isolating the bacteriome since it is small and fragile. Gently cut a hole on one side of the nymph and slightly press the other side to let out the bacteriomes. Then, mount a 20 microliter microloader onto a 0.5 to 10 microliter pipette, and extract the individual bacteriome from the PBS into the microloader.
Wash the bacteriome to eliminate the contamination of other whitefly tissues by pipeting the bacteriome into the PBS and extracting it. Repeat the wash three times. Repeating the wash is highly recommended to eliminate as much contamination as possible.
Immediately pipette the washed bacteriome into a centrifuge tube containing 60 microliters of PBS. Syringe filter the assembled bacteriome through a five micrometer filter membrane. Repeat the filtration multiple times to thoroughly move the mixed liquid through the membrane.
To amplify the filtrate, prepare buffer D2 to be used with the recommended protocol for amplification of genomic DNA from blood or cells with some modifications. Directly add 1.5 microliters of filtrate to a centrifuge tube followed by 1.5 microliters of buffer D2.Mix well and incubate the sample on ice for 10 minutes. Then add 1.5 microliters of stop solution.
Add 15 microliters of the reaction buffer and one microliter of DNA polymerase and gently mix. Incubate the mixture at 30 degrees Celsius for 16 hours followed by 65 degrees Celsius for three minutes. Carry out PCR directly on the amplified filtrate using specific primers designed for bacteria to confirm the bacteria species.
Also conduct PCR to confirm whether there is contamination from the host genome by using primers for the whitefly genes beta-actin and EF1 according to the previously published method. Finally, carry out meta genome sequencing according to the text protocol. In this figure, cotton for rearing whiteflies and several developmental stages of whiteflies are shown.
Including an adult and first, second, and fourth instar nymphs. FISH analysis of the endosymbionts Portiera and Hamiltonella within the fourth instar nymph of MEAM1 is shown here. Overlapping of the two bacteria is observed and both are confined to the bacteriocytes of the whitefly.
Shown here are the transmission electron microscopy images of the Portiera endosymbiont of the whitefly indicating that Portiera may lose its cell wall. After sequencing and cleaning up the contamination data for adapters and duplications the complete genome of the obligate symbiont Portiera was acquired. Resulting in a circular genome of 358, 232 base pairs.
In addition, a draft genome of Hamiltonella was obtained that included 138 contigs that was assembled to 89 scaffolds based on paired end relationships. Once master this technique can be done in 20 hours if it is performed properly. While attempting this procedure, it's important to remember to keep the samples from the contamination of the environmental bacteria.
Following this procedure, other analysis like RA sequence and the mass bacteriome can be performed in order to answer additional questions like endosymbiont gene expression and metabolism. After its development, this technique paved the way for the researchers in the field of entomology and microbiology to explore the function of endosymbionts in insects or arthropods. After watching this video, you should have a good understanding of how to extract symbionts from insect bacteriomes.
Don't forget that working with some of the reagents can be hazardous and the precautions like wearing gloves should always be taken while performing this procedure.
