This work was supported by National Key Research and Development Program (Grant number: 2017YFD0200600), the earmarked fund for China Agriculture Research System (grant number: CARS-23-D07) and the Bill &#38; Melinda Gates Foundation (Investment ID OPP1149777). We thank Prof. Jian-Xiang Wu for providing TYLCV CP antibodies.

1. Whitefly, Virus, Plants, and Acquisition of the Virus
<ol>
	<li>Rear whiteflies (MEAM1) on cotton (Gossypium hirsutum cv. Zhemian 1793) in insect-proof cages in a greenhouse at 26 &#177; 1 &#176;C with a 14:10 light:dark cycle and 60 &#177; 10% relative humidity.</li>
	<li>Perform conventional PCR based on whitefly mitochondrial cytochrome oxidase I gene to determine the purity of the whitefly population.
	<ol>
		<li>Collect 20 adult whiteflies and transfer them individually to a PCR tube containing 30 &#956;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>Add 5&#8211;7 ceramic beads (2 mm in diameter) into each PCR tube and grind the samples to perform the tissue lysis.</li>
		<li>Incubate all samples at 65 &#176;C for 1 h followed by 10 min at 100 &#176;C, and then centrifuge briefly. Use this supernatant as the template for PCR amplification. 
		NOTE: The incubation time at 65 &#176;C can be increased if necessary.</li>
		<li>Prepare the PCR reaction as follows: 1 U of Taq DNA polymerase, 2 &#181;L of 10x buffer (with Mg2+), 1.6 &#181;L of dNTP mixture (2.5 mM), 0.5 &#181;L of each primer (10 &#956;M each), 2 &#181;L of whitefly tissue lysate supernatant, and double distilled water to a total volume of 20 &#181;L per reaction. The forward primer sequence is 5&#39;-TTGATTTTTTGGTCATCCAGAAGT-3&#39; and the reverse primer sequence is 5&#39;-TAATATGGCAGATTAGTGCATTGGA-3&#39;. 
		NOTE: A negative control where the DNA is substituted with nuclease-free water and a positive control with previously verified MEAM1 whitefly DNA should be included.</li>
		<li>Perform PCR using the following cycling parameters: initial denaturation at 95 &#176;C for 3 min, followed by 35 cycles of denaturation at 95 &#176;C for 30 s, annealing at 50&#8211;55 &#176;C for 30 s, and extension at 72 &#176;C for 1 min, followed by 72 &#176;C for 10 min.</li>
		<li>Digest the amplified product with restriction enzyme Taq I. To do so, prepare the digestion mixture with 0.1 &#181;L (1 U) of enzyme,1 &#956;L of 10x Taq I Buffer, 1 &#956;L of BSA, 5 &#956;L of amplified product, and double distilled water to a total volume of 10 &#956;L. Incubate at 65 &#176;C for 1 h in a thermocycler.</li>
		<li>Perform agarose gel electrophoresis of the samples by loading 5&#8211;7 &#181;L of each digested product and a DNA ladder (100-2,000 bp) into the wells of a 1% agarose gel. Then, observe the DNA band sizes by gel-red staining in a gel documentation machine using UV light. Compare the band sizes of the digested products to the positive control to determine the purity of the whitefly population.</li>
	</ol>
	</li>
	<li>Agro-inoculate the infectious clone of TYLCV (GenBank accession number AM282874.1) (pBINPLUS-SH2-1.4A) into 3&#8211;4 true leaf stage tomato plants (Solanum lycopersicum L. cv. Hezuo 903).
	<ol>
		<li>Inoculate 10 mL of Luria&#8211;Bertani (LB) medium containing kanamycin (50 &#956;g/mL) and rifampicin (50 &#956;g/mL) with a single colony. Incubate at 28 &#176;C with shaking at 200 rpm until the OD600 reaches ~1.5.</li>
		<li>Harvest agrobacteria by centrifugation at 4,000 x g for 10 min. Discard the supernatant.</li>
		<li>Resuspend pellets in 10 mL of infiltration buffer, consisting of 200 &#956;M acetosyringone, 10 mM 2-(N-morpholino) ethane sulfonic acid (MES), and 10 mM MgCl2. Incubate at room temperature (RT) for 1&#8211;2 h.</li>
		<li>Infiltrate 2&#8211;3 plant leaves using the mixture from step 1.3.3 with a 1 mL needleless syringe. Maintain plants in a greenhouse at 26 &#177; 1 &#176;C.</li>
	</ol>
	</li>
	<li>About 1 month later, perform a visual inspection and choose plants showing yellow curl leaf and stunt symptoms.</li>
	<li>Transfer non-viruliferous whitefly adults onto TYLCV-infected tomato plants for 48 h for virus acquisition.</li>
</ol>
2. Dissection and Collection of Midguts, Primary Salivary Glands (PSG), Ovaries, and Hemolymph of Female Whiteflies
<ol>
	<li>Collect whiteflies using aspiration, and then place them on ice to put them in a coma. Add 1x PBS (phosphate-buffered saline) buffer onto a microscope slide, and then place whiteflies in the 1x PBS buffer for dissection under a stereo microscope.
	<ol>
		<li>For the midgut dissection, break the abdomen of the whitefly and pull out the midgut using a fine acupuncture needle (0.25 mm in diameter). Transfer the midgut to clean, 1x PBS.</li>
		<li>For PSG dissection, break the body of the whitefly in the thorax near the head from the dorsal side. Next, insert a needle into the thorax to fix the whitefly, and use another needle to shake the whitefly until the two salivary glands are separated from the body. Then, transfer the PSGs to clean, 1x PBS.</li>
		<li>For the ovary dissection, completely break the abdomen of the whitefly, and use a needle to separate the ovaries from other tissues. Then, remove excess tissues by pipetting.</li>
		<li>For the hemolymph collection, add 10 &#956;L of 1x PBS buffer onto a microscope slide, and then place a whitefly in the buffer. To obtain the hemolymph, gently tear a hole in the abdomen using a fine acupuncture needle, and slightly press the abdomen to release the hemolymph out into the buffer. Collect 8 &#956;L of the liquid as the hemolymph sample.</li>
	</ol>
	</li>
</ol>
3. Localization of TYLCV in Whitefly Midgut, Primary Salivary Glands, and Ovaries by Immunofluorescence
NOTE: The procedure for the localization of TYLCV in the midguts is presented as an example. The method can be used for PSGs and ovaries.
<ol>
	<li>Dissect the midguts in TBS buffer (10 mM Tris-HCl, 150 mM sodium chloride, pH = 7.5) as described in step 2.2.1. Collect 15&#8211;20 whitefly midguts, transfer them into a glass Petri dish (3.5 cm in diameter), and then remove excess TBS buffer.</li>
	<li>Add 1 mL of 4% paraformaldehyde to fix midguts for 2 h at room temperature, and then remove the fixative solution. Pipette slowly to wash the midguts 3x in TBS for 2 min each.</li>
	<li>Incubate the midguts in 1 mL of 0.5% Triton X-100 for 30 min to permeabilize. Then remove the permeabilization solution and wash the midguts 3x with TBST (1x TBS with 0.05% Tween 20) as described in step 3.2.</li>
	<li>Add 1 mL of 1% BSA (bovine serum albumin prepared in TBST) to block the midguts. Perform the blocking step at RT for 2 h, and then remove the blocking solution and wash the midguts 3x in TBST.</li>
	<li>Dilute the primary antibodies (MAb 1C4 against TYLCV coat protein) at 1:400 with TBST24 and add the solution into the Petri dish to immerse the midguts. Incubate at 4 &#176;C overnight in the dark. Discard the primary antibody solution and wash 3x in TBST.</li>
	<li>Dilute the secondary antibodies (anti-mouse) at 1:400 with TBST and add the solution into the Petri dish to immerse the midguts. Incubate the dish for 2 h at RT in the dark. Discard the secondary antibody solution and wash the midguts 3x in TBST.</li>
	<li>Transfer the midguts to a clear microscope slide. Aspirate as much TBST off the slide as possible. Add 1 drop of DAPI (4&#39;, 6-diamidino-2-phenylindole, dihydrochloride) to stain the nucleus, and then place the coverslip and seal with nail polish. Examine under a confocal microscope.</li>
</ol>
4. Quantification of TYLCV in Whitefly Midgut, Primary Salivary Glands, Hemolymph, and Ovaries with qPCR
<ol>
	<li>Dissect the whitefly midguts, PSGs, hemolymph, and ovaries in 1x PBS as described above.</li>
	<li>Extract the DNA of the midguts, PSGs, hemolymph, and ovaries.
	<ol>
		<li>After dissection, wash the midguts, PSGs, and ovaries in clean, 1x PBS buffer 2&#8211;3x.</li>
		<li>Collect 20 midguts or 20 PSGs in 5 &#956;L of 1x PBS and transfer them into 25 &#956;L lysis solution. Transfer each ovary in 5 &#956;L of 1x PBS and then transfer into 25 &#956;L lysis solution individually.</li>
		<li>Grind midguts, PSGs, and ovaries samples as described in step 1.2.2.</li>
		<li>Collect 8 &#956;L of hemolymph with 1x PBS into a PCR tube, and then add 10 &#956;L of lysis solution. Mix thoroughly.</li>
	</ol>
	</li>
	<li>Extract DNA from all samples as described in 1.2.3.</li>
	<li>Prepare two separate qPCR master mixes (18 &#956;L each) for TYLCV and the internal control (&#946;-actin gene) as follows: 10 &#956;L of SYBR Green mix, 0.8 &#956;L of each primer (10 &#956;M each), and 6.4 &#956;L of double distilled water per reaction. For TYLCV, the forward primer sequence is 5&#8242;-GAAGCGACCAGGCGATATAA-3&#8242; and the reverse primer sequence is 5&#8242;-GGAACATCAGGGCTTCGATA-3&#8242;. For &#946;-actin, the forward primer sequence is 5&#8242;-TCTTCCAGCCATCCTTCTTG-3&#8242; and the reverse primer sequence is 5&#8242;-CGGTGATTTCCTTCTGCATT-3&#8242;.</li>
	<li>Dispense 18 &#181;L of the master mix into vials of qPCR strip tubes or a 96 well plate. Then, add 2 &#181;L of the DNA samples into each vial. Perform all reactions with at least three technical replicates and three biological replicates. 
	NOTE: Step 4.5 should be performed on ice.</li>
	<li>Close the qPCR tubes or seal the 96 well plates with adhesive plate seal.</li>
	<li>Centrifuge to collect the liquid at the bottom of the qPCR tubes or 96 well plate.</li>
	<li>Place the tubes or plate into the real-time thermal cycler and perform qPCR.
	<ol>
		<li>Perform the qPCR using the following cycling parameters: 95 &#176;C for 30 s, followed by 40 cycles of 95 &#176;C for 5 s and 60 &#176;C for 34 s for primer annealing and elongation, 1 cycle at 95 &#176;C for 15 s, ending with a melting curve step of 60 &#176;C to 95 &#176;C with an increment of 0.5 &#176;C per 15 s.</li>
		<li>Choose SYBR as the fluorophore dye and unknown as sample type.</li>
	</ol>
	</li>
	<li>Export the quantitative data to a spreadsheet format and analyze the relative quantity of viral DNA by the 2&#8722;&#916;&#916;CT method28.</li>
</ol>

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The authors have nothing to disclose.

Here we describe a protocol for the localization and quantification of a begomovirus in the tissues of its whitefly vector by immunofluorescence and qPCR. Dissection represents the first step to localize and quantify the virus in whitefly tissues. The whitefly body is about 1 mm in length, which means that the tissues are extremely small, and it is difficult to dissect them. Besides, there are strong connections between the tissues. For example, the ovaries are tightly connected with the bacteriocytes, making them diffic...

In the last decades, begomoviruses (genus Begomovirus, family Geminiviridae) have caused serious damage to the production of many vegetable, fiber, and ornamental crops worldwide1. Begomoviruses are transmitted in a persistent manner by the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), which is a complex species containing over 35 cryptic species2,3. Begomoviruses may directly or indirectly affect whitefly physiology and behavior, such as fecundity4, longevity4, and host preference5,6. Furthermore, the transmission efficiency of a given begomovirus species/strain varies for different whitefly cryptic species even under the same experimental conditions7,8,9,10, indicating that there is a complex interaction between begomoviruses and whiteflies. To better understand the mechanisms underlying whitefly-begomovirus interactions, localization and quantification of the virus in whitefly tissues are essential.
Tomato yellow leaf curl virus (TYLCV) is a begomovirus that was first reported in Israel but nowadays causes serious damage to tomato production worldwide11,12. Due to its economic importance, it is one of the best-studied begomoviruses13. Like other monopartite begomoviruses, TYLCV is a single-strand circular DNA virus with a genome size of about 2,800 nucleotides14. While still under debate, several lines of evidence support the replication of TYLCV in whiteflies15,16,17. Moreover, the interaction of TYLCV particles and whitefly proteins has been reported6,18,19,20. For virus transmission, whiteflies acquire TYLCV by feeding on virus-infected plants, virions pass along the food canal to reach the esophagus, penetrate the midgut wall to reach the hemolymph, and then translocate into the primary salivary glands (PSGs). Finally, virions are egested with saliva along the salivary duct into plant phloem21. Furthermore, several studies show that TYLCV is able to be transovarially transmitted from female whiteflies to their offspring22,23. In other words, to achieve productive transmission, the virus has to overcome cellular barriers within the whitefly to translocate from one tissue to another. During the crossing of these barriers, interactions between whitefly and virus proteins are likely to occur, probably determining the efficiency with which the viruses are transmitted.
Immunofluorescence is a commonly used technique for protein distribution analysis. The specificity of antibodies binding to their antigen form the basis of immunofluorescence. Due to the economic significance of TYLCV, monoclonal antibodies against the TYLCV coat protein have been developed, offering a highly sensitive way to localize the virus24. Quantitative PCR (qPCR) allows sensitive and specific quantification of nucleic acids. This technique is most frequently based on the use of hydrolysis probe (e.g., TaqMan) or fluorescent dye (e.g., SYBR Green) detection. For hydrolysis probe-based qPCR, specific probes are needed, which consequently increase the cost. Fluorescent dye-based qPCR is simpler and more cost-effective, because labeled amplicon-specific hybridization probes are not required25. So far, several studies have used immunofluorescence and qPCR along with other methods to investigate the complex begomovirus-whitefly interactions. For example, Pan et al. performed qPCR and immunofluorescence analysis of the virus in whitefly tissues and found that the difference in ability to transmit tobacco curly shoot virus (TbCSV) between whitefly species AsiaII 1 and Middle East Asia Minor 1 (MEAM1) was due to the virus being able to efficiently cross the midgut wall of AsiaII1 but not MEAM18. Similarly, while Mediterranean (MED) whiteflies can readily transmit TYLCV, they fail to transmit tomato yellow leaf curl China virus (TYLCCNV). Selective transmission was investigated using immunofluorescence detection of virus in the PSGs, which showed that TYLCCNV do not easily cross the PSGs of MED whiteflies26. Immunofluorescence colocalization of TYLCV CP and the autophagy marker protein ATG8-II in whitefly midguts shows that autophagy plays a critical role in repressing the infection of TYLCV in the whitefly27.
Here, using TYLCV as an example, we describe a protocol for the localization of begomoviruses in whitefly midguts, PSGs, and ovaries by an immunofluorescence technique. The technique includes dissection, fixation, and incubation with primary and dye-labeled secondary antibodies. Fluorescence signals showing the location of viral proteins in the whitefly tissues can then be detected under a confocal microscope. More importantly, this protocol can be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV using SYBR Green-based qPCR in whitefly midguts, PSGs, hemolymph, and ovaries, that can be used to compare the amount of virus in different whitefly tissue samples.

<table><tbody><tr><td>4% Paraformaldehyde</td><td>MultiSciences</td><td>F0001</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-ph&amp;eacute;nylindole (DAPI)</td><td>Abcam</td><td>ab104139</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>MultiSciences</td><td>A3828</td><td></td></tr><tr><td>CFX Connect Real-Time PCR Detection System</td><td>Bio-RAD</td><td>185-5201</td><td></td></tr><tr><td>Confocal microscopy</td><td>Zeiss</td><td>LSM800</td><td></td></tr><tr><td>Dylight 549-goat anti-mouse</td><td>Earthox</td><td>E032310-02</td><td>Secondary antibody</td></tr><tr><td>Monoclonal antibody (MAb 1C4)</td><td></td><td></td><td>Primary antibody</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sangon Biotech</td><td>B548119-0500</td><td></td></tr><tr><td>Stereo microscope</td><td>Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>TB green premix Ex Taq (Tli RNase H Plus)</td><td>TaKaRa</td><td>RR820A</td><td>qPCR master mix</td></tr><tr><td>Thermocycler</td><td>Thermofisher</td><td>A41182</td><td></td></tr><tr><td>Tissuelyzer</td><td>Shaghai jingxin</td><td>Tissuelyser-48</td><td></td></tr><tr><td>Triton-X-100</td><td>BBI life sciences</td><td>9002-93-1</td><td></td></tr><tr><td>Tween 20</td><td>BBI life sciences</td><td>9005-64-5</td><td></td></tr></tbody></table>

localization and quantification of begomoviruses in whitefly tissues by immunofluorescence and quantitative pcr

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

The MEAM1 whiteflies of the B. tabaci complex and TYLCV were used as an example here to describe the procedures. The overview of the immunofluorescence and viral quantification procedures described in this manuscript is shown in Figure 1. Figure 2 shows representative results of immunofluorescence detection of TYLCV and DAPI staining in PSGs, midguts, and ovaries, indicating that TYLCV accumulated more in the PSGs and midguts, and less in the ovaries. <...

Watch this Scientific Journal Video about Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR at JoVE.com

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...

localization-quantification-begomoviruses-whitefly-tissues

Research

JoVE Journal

Immunology and Infection

5.5K Views.  Zhejiang University. We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

Video: Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Genetics

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Environment

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13, ecology2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14 or compounds15, new cultural approaches1,4,19, or the selection and development of resistant cultivars7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.
Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.
There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.

Improving our understanding of and our ability to manage many of the insect-transmitted plant viruses requires the use of the vector. Insect transmission of plant viruses is a tritrophic interaction, and as such requires the manipulation of insects, virus, and plant. Vectors must be reared in large numbers and manipulated in such a way as to insure high rates of transmission to test plants. The basics of rearing and manipulating the whitefly, Bemisa tabaci (Hemiptera: Aleyrodidae), a vector of plant viruses belonging to several emerging plant virus genera, representing a large number of economically significant viruses is presented.

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several ...

Biology

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation of RNA virus transmission using Wolbachia-transfected mosquitoes. Key strategies to control viruses include the manipulation of host reproduction via cytoplasmic incompatibility and the inhibition of viral transcripts via immune priming and competition for host-derived resources. However, the underlying mechanisms of the responses of Wolbachia-transfected mosquitoes to viral infection are poorly understood. This paper presents a protocol for the in vitro identification of Wolbachia infection at the nucleic acid and protein levels in Aedes albopictus (Diptera: Culicidae) Aa23 cells to enhance the understanding of the interactions between Wolbachia and its insect vectors. Through the combined use of polymerase chain reaction (PCR), quantitative PCR, western blot, and immunological analytical methods, a standard morphologic protocol has been described for the detection of Wolbachia-infected cells that is more accurate than the use of a single method. This approach may also be applied to the detection of Wolbachia infection in other insect taxa.

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

Four methods were used to detect intracellular Wolbachia, which complemented each other and improved the detection accuracy of Wolbachia infection of Aedes albopictus-derived Aa23 and Aa23-T&#160;cured of native Wolbachia infection using antibiotics.

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

Virus Propagation and Cell-Based Colorimetric Quantification

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and potentially Guillain-Barr&#233; syndrome in adults. Research on ZIKV to understand the disease mechanisms is important to facilitate vaccine and treatment development. The method of quantifying viruses is crucial and fundamental in the field of virology. The focus forming assay (FFA) is a virus quantification assay that detects the viral antigen with antibodies and identifies the infection foci of cells using the peroxidase immunostaining technique. The current study describes the virus propagation and quantification protocol using both 24-well and 96-well (high throughput) formats. Compared with other similar studies, this protocol has further described foci size optimization, which can serve as a guide to expand the use of this assay for other viruses. Firstly, ZIKV propagation is performed in Vero cells for 3 days. The culture supernatant containing ZIKV is harvested and quantitated using the FFA. Briefly, the virus culture is inoculated onto Vero cells and incubated for 2-3 days. Foci formation is then determined after optimized staining processes, including cell fixation, permeabilization, blocking, antibody binding, and incubation with peroxidase substrate. The stained virus foci are visualized using a stereo microscope (manual counting in 24-well format) or software analyzer (automated counting in 96-well format). The FFA provides reproducible, relatively fast results (3-4 days) and is suitable to be used for different viruses, including non-plaque-forming viruses. Subsequently, this protocol is useful for the study of ZIKV infection and could be used to detect other clinically important viruses.

The present protocol describes the propagation of Zika virus (ZIKV) in Vero African green monkey kidney cells and the quantification of ZIKV using cell-based colorimetric immunodetection methods in 24-well and 96-well (high throughput) formats.

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and ...

Fluorescence In Situ Hybridization to Study Mosquito Spermatogenesis

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Author Spotlight: Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

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.

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.

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

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

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Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Transmitting Plant Viruses Using Whiteflies

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Virus Propagation and Cell-Based Colorimetric Quantification

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci