This work was supported by grants from the National Natural Science Foundation of China (31630058 to X.W. and 31772134 to W.L.).

1. Nonviruliferous insect rearing
<ol>
	<li>Collect WBPHs from rice fields and rear with rice seedlings in 1 L glass beakers covered with insect-proof net in an incubator at 28 &#176;C with 16 h light and 8 h dark. Because SRBSDV is not transmitted via eggs, newly hatched nymphs are not viruliferous.</li>
	<li>With a brush pen, gently brush insects from the beaker rearing insects into a new beaker of fresh rice seedlings each week until WBPH nymphs have hatched. Continue rearing these hatched nonviruliferous nymphs to ...

<ol>
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For best results, a few key points should be considered. First, a high ratio of viruliferous insects among the total population is necessary. Although the minimum AAP for SRBSDV by WBPH nymphs and adults is 5 min17, the insects should be allowed to feed on fresh SRBSDV-infected rice plants for 2 d to achieve an acquisition efficiency of up to 80%. Since the SRBSDV virions can be detected in 80% of the midguts18, we excised and labeled the viruliferous insects at 2 d after a...

Most described plant viruses are transmitted by insects from the order Hemiptera that includes aphids, whiteflies, leafhoppers, planthoppers, and thrips1,2. The piercing-sucking mouthparts of hemipteran insects pierce the plant tissue for feeding and secreting saliva, concomitantly efficiently transmitting the virus2. Different transmission mechanisms of plant viruses by vector insects have been described. These include nonpersistent, semipersistent and persistent. The persistent type is either non-propagative or propagative3,4, but for both of these types, the transmitted virus must move throughout the body of the insect. In the persistent-propagative mode, viruses initially infect and replicate in the epithelial cells of the insect&#39;s gut, then disseminate into different tissues, and eventually into the salivary glands, from where they can then be introduced into a plant through the saliva during insect feeding5,6. Persistent transmitted viruses move through different organs and replicate in their insect vectors, which requires specific interactions between virus and vector components at different stages7,8.
Viral proteins and insect proteins must interact to facilitate critical processes for virus recognition, infection, replication, or dissemination in the vector insects9,10. Although optical microscopy can be used to observe cellular structures in insects, it cannot show virion distribution, cellular localization or colocalization of viral proteins and insect protein, or the ultrastructure of insect tissues and cells. Immunofluorescence labeling was first performed by Coons et al. in the phagocytic cells of the mouse by means of labeling specific fluorescein antibodies, and now it is used widely11. The immunofluorescence technique, also known as the fluorescence antibody technique, is one of the earliest immunological labeling techniques developed and is based on the specific binding reaction between the antigen and the antibody11,12. The known antibody is first labeled with fluorescein, which is used as a probe to detect the corresponding antigens in the cells or tissues13,14. After the fluorescein-labeled antibody binds to the corresponding antigen in cells or tissues, the probe will emit bright fluorescence when irradiated with excitation wavelengths and viewed with a fluorescence microscope to localize the antigen15.
Most vector insects of plant viruses are hemipterans. A higher population density of vector insects that have a high transmission efficiency for the plant virus can lead to virus epidemics5. Southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), one of the most serious pathogens of rice, has rapidly spread throughout rice-growing areas in East and Southeast Asia, and caused serious yield losses since 201016,17. Adults and nymphs of the white backed planthopper (WBPH, Sogatella furcifera Horv&#225;th) transmit SRBSDV to rice in a persistent-propagative manner with high efficiency.Field studies have shown that outbreaks of SRBSDV-induced rice black streaked dwarf disease usually coincide with mass long-distance migration of WBPHs, a crucial factor in SRBSDV epidemics7,8,18. Vesicle-associated membrane protein 7 (VAMP7) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), which can mediate the transport of substances via vesicle fusion. VAMP7 interacts with the outer major capsid protein of SRBSDV in vitro, which indicates that VAMP7 might be closely associated with virus transmission16.
In the protocol presented here, we excised the gut from viruliferous WBPH as an example to label SRBSDV virions and VAMP7 in midgut epithelial cells16. As the initial invasion site of virus, the midgut epithelium plays vital roles in virus infection, replication, and transmission. First, we detailed the steps to excise the gut from nymphs and adults of&#160;WBPHs. Second, we used specific fluorescein-labeled antibodies to label SRBSDV virions and VAMP7 in gut epithelial cells. Then we observed epithelial cells and the cellular location of the virions and VAMP7 via a laser scanning confocal microscope. The results showed that SRBSDV virions and VAMP7 could colocalize in the cytoplasm of the midgut epithelial cells, suggesting that the specific function of VAMP7 might be related to dissemination of virions from midgut epithelial cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3% Bull serum albumin (BSA)</td>
			<td>Coolaber</td>
			<td>SL1331</td>
			<td>Dilute antibodies</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Solarbio</td>
			<td>YA0771-18*18mm</td>
			<td>For slide making</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Beitja</td>
			<td>XTL-7045B1</td>
			<td>For insect dissection</td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss LSM880</td>
			<td>Observe fluorescence signal</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Solarbio</td>
			<td>ZBP-7105</td>
			<td>For slide making</td>
		</tr>
		<tr>
			<td>Mounting medium with 4&#39;6-diamidino-2-phenylindole (DAPI)</td>
			<td>Abcam</td>
			<td>AB104139</td>
			<td>Label cell necleus</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>For tissues fixation</td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>&#160;Invitrogen</td>
			<td>A22284</td>
			<td>Label actin of midgut epithiels</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Amresco</td>
			<td>0290C484</td>
			<td>For tissues permeation</td>
		</tr>
		<tr>
			<td>Tweezers (5-SA)</td>
			<td>AsOne</td>
			<td>6-7905-40</td>
			<td>For insect dissection</td>
		</tr>
	</tbody>
</table>

immunofluorescent labeling of plant virus and insect vector proteins in hemipteran guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

Figure 1&#160;illustrates all steps in this protocol: insect rearing, virus acquisition, excision of the gut, immunofluorescent labeling, and making the slide.
Excised WBPH guts from adults were fixed in 4% (m/v) paraformaldehyde, permeabilized with 2% (v/v) Triton X-100, and then incubated with Dylight 633 phalloidin10,18. The laser scanning confocal micrograph in Figure 2 sh...

Watch this Scientific Journal Video about Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts at JoVE.com

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

This protocol can be used to study various movement in insects, functions of insect proteins, and interactions between virus and vector insect in vivo. This method is efficient and informative. The structures of the insect gut and the auxiliary cells are clearly visualized when viewed with the laser scanning confocal microscopy.Demonstrating the procedure will be Lu Zhang, PhD from our laboratory. To begin, transfer non-viruliferous insects from glass beakers onto fresh southern rice black streaked dwarf virus, SRBSDV, infected rice plants covered with an insect proof net for a two-day virus acquisition access period. Then collect the insects in glass beakers containing fresh rice seedlings.After two days, collect the insects from the glass beakers using a manual aspirator for dissection and excision of the gut. Prepare 0.01 molar PBS, 4%paraformaldehyde, and 2%Triton X-100 as described in the text manuscript. Use a pipetter to place 100 microliters of PBS on a glass slide and place the slide on the stage of an optical microscope.Use manual aspirator to collect the SRBSDV infected adults from the glass beakers and place them in a 1.5 milliliter tube on ice to paralyze the insects. Transfer a paralyzed adult into the 100 microliters of PBS on the slide with the abdomen up. Use tweezers to clamp the body and remove the head with another set of tweezers.With one set of tweezers, clamp the sides of the abdomen and clamp the ovipositor or the copulatory organ of the tail with the other set, then carefully pull the intersegmental membrane of one abdominal segment to slowly expose the gut in the abdomen. Continue tearing away the membrane and gradually pull out the complete gut from the abdomen. Gently pull off the tail which is connected to the end of the gut to remove the complete gut.Place excised guts into a 200 microliter centrifuge tube. Add 200 microliters of PBS to the tube and use a pipette to thoroughly wash the guts. Use a pipetter to place 100 microliters of PBS on a glass slide and place the slide on the stage of an optical microscope.Use manual aspirator to collect the SRBSDV infected nymphs from glass beakers and place them in 1.5 milliliter tube placed on ice to paralyze the insects. Transfer a paralyzed nymph into the 100 microliters of buffer on the slide with the abdomen facing up. Detach the tail of the nymph, then clamp the insect body to fix it gently and use the other pair to clamp the head.Gently pull the head away from the body while still maintaining its attachment to the gut so that the head is detached from the body, but the gut is still attached to the thorax and abdomen. With the tweezers still clamping the body, use the other pair to move the head carefully and gradually pull out the gut. Detach the gut from the head to obtain an intact gut without damaging the body.Place the excised guts into a tube. Add PBS to the tube and gently suck release the solution with a pipette to wash the guts thoroughly. Prepare the antibodies and mounting medium as described in the text manuscript.Place the freshly excised and PBS washed WBPH guts in 100 microliters of 4%paraformaldehyde in a 200 microliter centrifuge tube at room temperature. After two hours, replace the paraformaldehyde solution with 200 microliters of PBS. After 10 minutes, remove the PBS to eliminate any paraformaldehyde and repeat the PBS wash step twice.After two PBS washes, add 200 microliters of non-ionic detergent Triton X-100 to permeabilize the samples at room temperature. After 30 minutes, remove the Triton X-100 and wash away any remaining detergent with three 10-minute washes with PBS. Dilute the labeled antibodies one to 50 with 50 microliters of bull serum albumin.Add the diluted antibodies to the tube and incubate the samples overnight at four degrees Celsius. On the morning after removing the antibody, wash away the remaining antibody diluent with three 10-minute washes with PBS. Dilute one microliter of DyLight 633-Phalloidin with 50 microliters of PBS and add 50 microliters of diluted phalloidin to the tube for a two-hour incubation at room temperature.After removing phalloidin, thoroughly wash away the remaining phalloidin with three 10-minute washes with PBS. Place a drop of mounting medium containing DAPI on a microscope slide. Transfer the guts to the medium and gently place the cover glass over the sample without creating bubbles.The representative laser scanning confocal micrograph of excised WBPH guts from adults after labeling with phalloidin showed three sections:the foregut, midgut, and hindgut. Among these, midgut is the initial infection site of SRBSDV. The monolayer epithelial cell structure of the gut facilitates the study of the cellular localization of insect proteins and colocalization of viral and insect proteins.Excised WBPH guts with DyLight 488 labeled anti-VAMP-7 and SRBSDV virions with DyLight 549 labeled anti-SRBSDV antibody are shown here. VAMP-7 and SRBSDV virions were shown to co-localize in the cytoplasm suggesting that VAMP-7 may play a role in virus transmission in vivo. Permeabilized guts should be cleaned thoroughly after incubation with luciferin conjugated antibodies to reduce the background and non-specific binding.This is a reliable method to view the localization of virus, other pathogens, and insect proteins in insects. It provides the foundation for further studies of the relationship between pathogens and insects.

immunofluorescent-labeling-plant-virus-insect-vector-proteins

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

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