Financial support for this study was provided by the National Key Research and Development Program (2016YFC1200601) and the National Natural Science Foundation of China (31390421).

1. Whitefly Rearing and Cryptic Species Identification
<ol>
	<li>Maintain the whitefly species on cotton Gossypium hirsutum (Malvaceae) (cv. Zhe-Mian 1973) in cages under standard conditions of 27 &#177; 1 &#176;C, 70 &#177; 10% humidity and 14 h light: 10 h dark regime.</li>
	<li>Collect an individual adult whitefly and homogenize in 30 &#181;L of lysis buffer (10 mM Tris, pH 8.4, 50 mM KCl, 0.45% [wt/vol] Tween-20, 0.2% [wt/vol] gelatin, 0.45% [vol/vol] Nonidet P 40, 60 g/mL Proteinase K).</li>
	<li>Incubate...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+secondary+endosymbionts+among+different+putative+species+of+the+whitefly+Bemisia+tabaci.">Diversity of secondary endosymbionts among different putative species of the whitefly Bemisia tabaci.</a> Insect Sci. 20 (2), 194-206 (2013).</li><li>Bing, X. L., Yang, J., Zchori-Fein, E., Wang, X. W., Liu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+newly+discovered+symbiont+of+the+whitefly+Bemisia+tabaci+(Hemiptera:+Aleyrodidae).">Characterization of a newly discovered symbiont of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae).</a> Appl. Environ. Microbiol. 79 (2), 569-575 (2013).</li><li>Kliot, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridizations+(FISH)+for+the+localization+of+viruses+and+endosymbiotic+bacteria+in+plant+and+insect+tissues.">Fluorescence in situ hybridizations (FISH) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues.</a> J. Vis. Exp. (84), e51030 (2014).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J. Vis. Exp. (62), e3923 (2012).</li><li>Rao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+reduction+and+potential+metabolic+complementation+of+the+dual+endosymbionts+in+the+whitefly+Bemisia+tabaci.">Genome reduction and potential metabolic complementation of the dual endosymbionts in the whitefly Bemisia tabaci.</a> BMC Genomics. 16 (1), 226 (2015).</li><li>Thao, L. L., Baumann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+relationships+of+primary+prokaryotic+endosymbionts+of+whiteflies+and+their+hosts.">Evolutionary relationships of primary prokaryotic endosymbionts of whiteflies and their hosts.</a> Appl. Environ. Microbiol. 70 (6), 3401-3406 (2004).</li><li>Zhu, D. 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Since the endosymbionts within whiteflies cannot be cultured in vitro, dissection and assembling bacteriocytes is an effective way to obtain enough genetic material of endosymbionts. Before dissection, the species of whitefly and endosymbionts involved should be explicitly confirmed. The whitefly B. tabaci is a species complex with more than 35 morphologically indistinguishable species and different cryptic species may contain different endosymbionts. Portiera is uniformly harbored as an obliga...

Bacteria forming an intimate symbiotic relationship with relative hosts are widespread in arthropods1. The endosymbionts have been demonstrated to affect aspects of hosts, such as nutrition metabolism, reproduction, responses to environmental stresses2,3,4 etc., in almost every developmental stage5. However, the mechanism underpinning the associations still remains largely unknown. Genomics is of priority and importance when studying the potential functions and roles of bacteria. Some fundamental information, i.e. the taxonomic status, functional genes, metabolism pathways, secretion systems, can be inferred from genome sequences, which sheds lights on the potential roles of symbionts in symbiosis. With the development of high-throughput sequencing, a vast number of bacterial genomes have been sequenced with diverse functions revealed6.
Endosymbionts are of vital importance in hemipterans, such as aphids7, bedbugs8, psyllids9, brown planthoppers10 and cicadas11. For instance, Buchnera in aphids, as the obligate symbiont, has been demonstrated to be involved in essential amino acids biosynthesis, along with the genes from aphid genome12. Furthermore, transcriptional regulation of Buchnera is also revealed13. In psyllids, Carsonella is sequenced and ranked the smallest bacterial genome ever found14. All these hallmarks of endosymbionts are based and inferred from the genome sequences. Because these endosymbionts cannot be cultured in vitro, several approaches have been applied to isolate adequate bacteria for sequencing. In aphids, endosymbionts are extracted through centrifugation and filtration, and subjected to further genomic and transcriptomic analysis5. In brown planthoppers, endosymbionts are sequenced along with the whole insect genome10.
Whitefly B. tabaci is a species complex containing more than 35 morphologically indistinguishable species (cryptic species), among which, two invasive species have invaded all over the world and caused tremendous harm to agricultural production15. Of note, endosymbionts within the B. tabaci species have shown importance in the development of the pests16. To date, eight endosymbionts have been identified in the whitefly, including the obligate symbiont, Candidatus Portiera aleyrodidarum, and seven secondary symbionts Hamiltonella, Rickettsia, Arsenophonus, Cardinium, Wolbachia, Fritschea and Hemipteriphilus defined17,18.
Unlike the hemipterans described previously, the whitefly B. tabaci is an extremely tiny insect only 1 mm in length. Most endosymbionts are confined to bacteriocytes19 (specialized cells containing symbionts, which further form bacteriome in B. tabaci). In addition, these endosymbionts cannot be cultured in vitro. The only way to obtain endosymbionts from B. tabaci is to dissect the bacteriome out. However, there is difficulty in the dissection. First, the fragile bacteriome always links with other tissues of the whitefly, which is hard to separate. Secondly, the tiny size of the whitefly limits the isolation of enough bacteriome. Thirdly, endosymbionts cluster in the bacteriome, making it extremely complicated to acquire a single species of bacterium.
Here, we report a simple and inexpensive protocol to isolate whitefly endosymbionts for subsequent metagenome sequencing. Through dissection, purification and amplification, adequate endosymbiont DNA could be obtained and the species of bacteria could be confirmed. The described protocol can be used similarly in other arthropods.

<table border="0" cellpadding="0" cellspacing="0" width="775">
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Taq DNA polymerase</td>
			<td style="width:107px;">Takara</td>
			<td style="width:129px;">R001A</td>
			<td style="width:296px;">including rTaq, 10&#215;Buffer and dNTP</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Gel DNA extraction kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:129px;">28704</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">DNA sample sequencing system</td>
			<td style="width:107px;">ABI</td>
			<td style="width:129px;">ABI-3730XL</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Microtome</td>
			<td style="width:107px;">Leica</td>
			<td style="width:129px;">EM UC7</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Transmission electron microscopy</td>
			<td style="width:107px;">Hitachi</td>
			<td style="width:129px;">H-7650 TEM</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Stereo microscope</td>
			<td style="width:107px;">Zeiss</td>
			<td style="width:129px;">Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">20 &#956;L microloader</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:129px;">F2771951</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Filter holder</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:129px;">SX0001300</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Filter membrane filter</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:129px;">SMWP001300</td>
			<td>5.0 &#956;m SMWP</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">REPLI-g UltraFast Mini Kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:129px;">150033</td>
			<td>DNA amlification kit</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">NanoDrop</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:129px;">NanoDrop 2000</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Qubit Fluorometer</td>
			<td style="width:107px;">Thermo Fisher Scientific</td>
			<td style="width:129px;">Q33216</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Genome Sequencer</td>
			<td style="width:107px;">Illumina</td>
			<td style="width:129px;">Hiseq 2000</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for the extraction of endosymbionts from the whitefly bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

The Middle East Asia Minor 1 (MEAM1) species of the B. tabaci complex was taken as an example here for description. Cotton for rearing whiteflies and several developmental stages of whiteflies are shown in Figure 1 including a cotton plant, adult whitefly and the 1st, 2nd and 4th instar nymphs of whitefly (the 3rd instar nymph looks similarly as the 4th instar nymph). It was obvious that the 4<su...

Watch this Scientific Journal Video about Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci at JoVE.com

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

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.

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JoVE Journal

Biology

9.7K Views.  Zhejiang University. 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 isolati...