1. Whitefly Colony Maintenance
<ol>
	<li>Environmental Conditions: Whitefly colonies should be maintained in a controlled growth room. Control of relative humidity, temperature, photoperiod, and light intensity are essential for optimal colony growth (Figure 1). A temperature of 28 &#176;C, 30-50% relative humidity, and a 14 hr photoperiod will yield a colony that develops from egg to adult emergence in 18 days (this time varies with the plant host). Relative humidity should be kept below 70% to discourage the growth of insect and plant fungal pathogens. Fertilizer rates and watering must be reduced to discourage fungal pathogens and salt accumulation. The light intensity for the colony should be fairly high. VHO fluorescent bulbs used in sufficient numbers to generate approximately 800-1,000 ft candles at the canopy height are adequate for most plants (i.e. cotton, common bean, lima bean, tomato). Cleanliness is essential in a whitefly colony to maintain optimal rearing conditions.</li>
	<li>Colony Cages: Whiteflies should be maintained on plants in cages, rather than free in the growth room. Cages can be constructed of various materials but must allow for: ventilation, ease of access, ability to prevent whitefly escape or infiltration, and of sufficient size to maintain enough plants to generate the whitefly population needed. Good results have been obtained with organza plus aluminum screening (for strength) as well as whitefly-proof screening (see table of specific equipment and supplies).</li>
	<li>Colony Plant Preparation: The species of plant selected is an important consideration when starting a whitefly colony. The plant should be able to support a high insect population without collapsing. The plant chosen for a nonviruliferous colony should be a nonhost for the virus or viruses that are intended to be transmitted to avoid contamination in the colony and if maintaining a viruliferous colony, it must be host of the virus. The plants should grow fairly quickly to support emergence of new adults within 18 days of being introduced to the colony but not so quickly as to outgrow the cages within four weeks. Dwarf, bush, or patio-type plant cultivars are recommended as they often produce a similar leaf area to taller cultivars but without the stem elongation.</li>
	<li>It is very important to rear plants for the colony in greenhouses in cages that exclude whiteflies and other insects. Use of plants that are infested with whiteflies, thrips, or mites before they enter the colony can cause the colony to collapse. Infested plants also have the potential to be infected with insect-borne viruses which will interfere with virus transmission studies.</li>
</ol>
2. Whitefly Colony Establishment
<ol>
	<li>Start a colony for the first time using clean whiteflies - ones free of plant viruses, other insects, and insect pathogens. These can be obtained from collaborators or from the field. If field collected, whiteflies should be reared for at least 8 weeks on nonhost plants of the plant virus, and check for absence of plant symptoms to be sure they are free of plant viruses.</li>
	<li>Week 1: Introduce clean whiteflies to the first cage of plants. Whiteflies can be introduced by aspiration of known numbers or by gently shaking whiteflies from another source plant depending on the demands on the colony. During week 1, whiteflies are laying eggs on the underside of plant leaf surfaces. An approximate population size can be predicted for each emergence based on the number of adults used to lay eggs and the average number of eggs laid by the female whitefly on the host plant in question.</li>
	<li>Week 2: Begin another cage by introducing whiteflies to new host plants to lay eggs. The plants in the cage started in week 1 will have eggs, and some immature whiteflies covering the undersides of the leaves (Figure 2). Some adult whiteflies introduced during week 1 will still be alive.</li>
	<li>Week 3: Begin a third cage by introducing whiteflies to new plants to lay eggs. The plants in the cage started in week 1 will have immature as well as many new adult whiteflies emerging and there should be a very noticeable increase in the adult whitefly population. The plants in the cage started in week 2 will have many eggs and immature whiteflies covering the undersides of the leaves.</li>
	<li>Week 4: Begin a fourth cage by introducing whiteflies to new plants to lay eggs. The plants in the cage started in week 1 will have many adult whiteflies that are approximately one week post emergence. The plants in the cage started in week 2 will have many new adult whiteflies emerging and there should be a very noticeable increase in whitefly population. The plants in the cage started during week 3 will have many eggs and immature whiteflies covering the undersides of the leaves.</li>
	<li>Weekly after the first 4 weeks: Each week, start a new cage by introducing the adult whiteflies from the fourth oldest cage (the cage started in week 1) onto new plants in a new, clean cage. The old cage should be removed and its plants discarded. These whiteflies are approximately 1 week post-emergence. In the case of the viruliferous colony, these whiteflies will transmit virus to the new plants as well as lay eggs for the next generation of whiteflies.</li>
	<li>Plan transmission experiments to use the whiteflies that will emerge in the third week&#39;s cage. If more whiteflies are needed, plant numbers can be increased and more whiteflies can be added to the week 1 cage to increase whitefly population.</li>
</ol>
3. Method for Inoculation of Test Plants
<ol>
	<li>To insure high transmission rates: 1) Whiteflies must be handled as gently as possible to prevent damage to the insect which will reduce transmission rates; 2) There must be adequate leaf area available for whiteflies to either probe or feed. An increase in acquisition time or an increase in the number of acquisition host plants may increase low rates of infectivity in test plants due to crowding in either the acquisition or inoculation access period.</li>
	<li>Collection of Whiteflies. The following is a procedure for the collection and movement of precise numbers of whiteflies required for experimental purposes. To move large numbers of whiteflies, as is needed for resistance screening for example, it is only necessary to gently shake the whitefly-infested plants over the test plants or the virus-infected acquisition hosts (and later the test plants). Be sure to shake the plants over many locations to minimize aggregation effects.</li>
	<li>Newly emerged adult whiteflies (1-3 days post emergence) are highly active and feed often so tend to give the highest transmission rates 4. Older whiteflies still transmit but at a lower frequency. Multiple whiteflies per test plant (15-40 per plant) should be used for high rates of transmission since a ratio of 1 whitefly per plant often results in unacceptably low transmission rates3,4. The number needed depend upon the virus and the species of the acquisition and test plants.</li>
	<li>Assemble aspiration devices and collection vials. (Figure 3)</li>
	<li>With one hand, hold a yellow plastic card inside the colony cage that contains the whiteflies to be collected. Gently tap the plants to encourage the adult whiteflies to fly. Whiteflies will be attracted to the yellow card and will fly from the plant to the card where they can be collected using an aspirator and a very gentle breath. Never aspirate whiteflies that are feeding on plants. Their stylets are embedded in the plant while feeding so pulling them off the plants will break their stylets and rendering them unable to either acquire or transmit virus (Figure 4).</li>
	<li>Collect about 20 adult whiteflies into a single collection vial to minimize physical damage to the insects from repeated aspirations (Figure 5).</li>
	<li>To change the collection vial, gently tap the vial on a hard surface and cap with Parafilm while the whiteflies are disoriented from the tapping.</li>
	<li>Put a new collect vial on the aspirator and repeat until the number of whiteflies needed are collected. Whiteflies can remain in the collection vials at room temperature for several hours.</li>
	<li>Acquisition. Whiteflies are placed on a virus infected plant and allowed to feed on the infected plants for 48-72 hr. Acquisition periods longer than 72 hr generally do not increase transmission rates. For those viruses transmitted in a nonpersistent or semipersistent manner, 1 hr and several hours, respectively, are sufficient acquisition access periods (Table 1). Best results are obtained when whiteflies are given free access to plants in cages that confine the insects to a single plant or to multiple plants. However some experiments require acquisition from specific leaves in which case clip cages can be used. When using clip cages it is important to accommodate the whitefly feeding preference for the undersides of leaves. Also, crowding of whiteflies in clip cages can reduce transmission rates - acceptable rates of transmission have been achieved using 10 female whiteflies per clip cage of 2.5 cm diameter.</li>
	<li>Inoculation. Prepare a cage appropriate for the size of the inoculation and place the test plants inside. If only one plant is to be inoculated consider a single plant cage. If more than one plant is to be inoculated consider a small PVC&#160;frame/organza bag cage or an aluminum cage (Figure 6). The size of the cage used for inoculation should be slightly larger the size of the plant(s) to be inoculated. Higher transmission rates are obtained when whiteflies are kept close to the plant canopy and not given a lot of free space.</li>
	<li>Collection of whiteflies from acquisition hosts depends on the needs of the experiment. If small numbers are needed, aspirate whiteflies as described above. Place the collection vials of whiteflies inside the cage near the plants and remove the lid (Figure 7). Open the vial and release the whiteflies or invert and gently tap the vial to release the whiteflies. If large numbers are needed simply move acquisition plants with whiteflies into the cages where the test plants are located and gently shake the whiteflies off the acquisition host plant. For both types of introduction, be sure to distribute the whiteflies across the plants to minimize aggregation and insure a uniform inoculation.</li>
	<li>Allow the whiteflies to probe or feed for the appropriate amount of time, again dependent on the virus and to some extent the host plant (Table 1).</li>
	<li>Check the whiteflies at least once during inoculation access period to insure that whiteflies are probing or feeding by opening the cage and turning over some leaves on each plant.</li>
	<li>For the longer inoculation access periods, disturb the plants gently (using a bamboo stick or equivalent) to brush the tops of the plants and encourage the whiteflies to redistribute on the plants. Redistribution of whiteflies helps ensure a higher transmission rate by countering the natural tendency of whiteflies to aggregate.</li>
	<li>Termination. The inoculation access period is ended by killing the whiteflies with approved chemicals. Apply two insecticides one after the other: a contact insecticide to quickly terminate adult whiteflies and a systemic insecticide to terminate any whiteflies that develop in the following weeks and those missed by the contact insecticide (Figure 8). A contact insecticide such as insecticidal soap and systemic insecticides such as imidacloprid or pymetrozine have been used with good success.</li>
</ol>

<ol>
	<li>Antignus, Y., Mor, N., Ben Joseph, R., Lapidot, M., Cohen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV+absorbing+plastic+sheets+protect+crops+from+insect+pests+and+from+virus+diseases+vectored+by+insects.">UV absorbing plastic sheets protect crops from insect pests and from virus diseases vectored by insects.</a> Environ. Entomol. 25, 919-924 (1996).</li><li>Cohen, S., Gerling, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+whitefly-transmitted+viruses.">Epidemiology of whitefly-transmitted viruses.</a> Whiteflies: Their Bionomics, Pest Status and Management. , 211-225 (1990).</li><li>Cohen, S., Antignus, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+yellow+leaf+curl+virus,+a+whitefly-borne+geminivirus+of+tomatoes.">Tomato yellow leaf curl virus, a whitefly-borne geminivirus of tomatoes.</a> Adv. Dis. Vector Res. 10, 259-288 (1994).</li><li>Csizinszky, A., Schuster, D. J., Polston, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+UV-reflective+mulches+on+tomato+yields+and+on+the+silverleaf+whitefly.">Effect of UV-reflective mulches on tomato yields and on the silverleaf whitefly.</a> HortSci. 34, 911-914 (1999).</li><li>De Barro, P. J., Liu, S. -. S., Boykin, L. M., Dinsdale, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bemisia+tabaci:+A+statement+of+species+status.">Bemisia tabaci: A statement of species status.</a> Annu. Rev. Entomol. 56, 1-19 (2011).</li><li>Duffus, J. E., Gerling, D., Mayer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whitefly-borne+viruses.">Whitefly-borne viruses.</a> Bemisia: 1995 Taxonomy, Biology, Damage, Control and Management. , 255-288 (1996).</li><li>Lapidot, M., Czosnek, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+TYLCV-resistant+plants+using+whitefly-mediated+inoculation.">Screening for TYLCV-resistant plants using whitefly-mediated inoculation.</a> Tomato Yellow Leaf Curl Virus Disease. , 329-342 (2007).</li><li>Lapidot, M., Friedmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+for+resistance+to+whitefly-transmitted+geminviruses.">Breeding for resistance to whitefly-transmitted geminviruses.</a> Ann. Appl. Biol. 140, 109-117 (2002).</li><li>Lapidot, M., Polston, J. E., Stansly, P. A., Naranjo, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+epidemiology+of+Bemisia-vectored+viruses.">Biology and epidemiology of Bemisia-vectored viruses.</a> Bemisia: Bionomics and Management of a Global Pest. , 227-231 (2010).</li><li>Navas-Castillo, J., Fiallo-Olive, E., Sanchez-Campos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+virus+diseases+transmitted+by+whiteflies.">Emerging virus diseases transmitted by whiteflies.</a> Annu. Rev. Phytopathol. 49, 219-248 (2011).</li><li>Ng, J. C. K., Falk, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-vector+interactions+mediating+nonpersistent+and+semipersistent+transmission+of+plant+viruses.">Virus-vector interactions mediating nonpersistent and semipersistent transmission of plant viruses.</a> Ann. Rev. Phytopathol. 44, 212-21 (2006).</li><li>Polston, J. E., Cohen, L., Sherwood, T. A., Ben-Joseph, R., Lapidot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsicum+species:+Symptomless+hosts+and+reservoirs+of+Tomato+yellow+leaf+curl+virus+(TYLCV).">Capsicum species: Symptomless hosts and reservoirs of Tomato yellow leaf curl virus (TYLCV).</a> Phytopathology. 96, 447-452 (2006).</li><li>Polston, J. E., Hiebert, E., McGovern, R. J., Stansly, P. A., Schuster, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+range+of+Tomato+mottle+virus,+a+new+geminivirus+infecting+tomato+in+Florida.">Host range of Tomato mottle virus, a new geminivirus infecting tomato in Florida.</a> Plant Dis. 77, 1181-1184 (1993).</li><li>Polston, J. E., Sherwood, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pymetrozine+interferes+with+transmission+of+Tomato+yellow+leaf+curl+virus+by+the+whitefly+Bemisia+tabaci+(Hemiptera:+Aleyrodidae.">Pymetrozine interferes with transmission of Tomato yellow leaf curl virus by the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae.</a> Phytoparasitica. 31, 490-498 (2003).</li><li>Schuster, D. J., Thompson, S., Ortega, L. D., Polston, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+evaluation+of+products+to+reduce+settling+of+sweetpotato+whitefly+adults.">Laboratory evaluation of products to reduce settling of sweetpotato whitefly adults.</a> J. Econ. Ento. 102, 1482-1489 (2009).</li><li>King, A. M. Q., Adams, M. J., Carstons, E. B., Lefkowitz, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. , (2012).</li><li>Wintermantel, W. M., Stansly, P. A., Naranjo, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+efficiency+and+epidemiology+of+Criniviruses.">Transmission efficiency and epidemiology of Criniviruses.</a> Bemisia: Bionomics and Management of a Global Pest. , 319-332 (2010).</li><li>Yang, Y., Sherwood, T. A., Patte, C. P., Hiebert, E., Polston, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Tomato+yellow+leaf+curl+virus+(TYLCV)+Rep+gene+sequences+to+engineer+TYLCV+resistance+in+tomato.">Use of Tomato yellow leaf curl virus (TYLCV) Rep gene sequences to engineer TYLCV resistance in tomato.</a> Phytopathology. 94, 490-496 (2004).</li><li>Zehnder, G. W., Yao, C., Murphy, J. F., Sikora, E. R., Kloepper, J. W., Schuster, D. J., Polston, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbe-Induced+Resistance+Against+Pathogens+and+Herbivores:+Evidence+of+Effectiveness+in+Agriculture.">Microbe-Induced Resistance Against Pathogens and Herbivores: Evidence of Effectiveness in Agriculture.</a> Induced Plant Defenses Against Pathogens. , 335-355 (1999).</li></ol>

No conflicts of interest declared.

The methods described here were developed over a period of two decades and are based on the basic information provided by many studies of whitefly transmission, behavior, and biology. Since there are many publications, this manuscript refers primarily to reviews and to a few selected specific publications to illustrate the type of data used to develop this technique. It is very important in this procedure that the whiteflies are handled gently since breaking of legs and antennas can lead to abnormal behaviors and lower rates of transmission. Collection with vacuums and other mechanical suction devices has never produced good results. Another consideration for success is that the number of whiteflies used to obtain 100% transmission to test plants, must be determined for each combination of plant host and virus. This optimal ratio only needs to be established once. Best results have been obtained using young adult whiteflies (1-3 days past emergence) since they give the highest rates of transmission. Older adults can be used but larger numbers of whiteflies will be needed to compensate for their reduced rates of transmission. Females are known to transmit at higher rates than males, since they feed more often than males. However it is usually not worth the time to separate the sexes for transmission. This procedure can be modified in many ways to suit the resources and needs of the researcher.
When working with multiple viruses it is best to keep a single colony of whiteflies reared on nonhost plants of the virus. These whiteflies can be collected placed on virus-infected plants for acquisition and then after acquisition the whiteflies can be moved again to the test plants. Plants intended for the colony should be grown in greenhouses in cages to prevent the introduction of whiteflies that might infect them with another virus. Infestation of these plants with mites, thrips, and other plant pests can compromise the health of the colony and cause it to collapse. Finally watering of the colony plants has to be done with care. These plants are subject to extreme stress by the feeding of so many whiteflies and the lower than normal light conditions. Root rotting fungi which are introduced on the peat in the soilless mixes can become a problem if the plants are overwatered even once. Pretreatment of the plants with a fungicide drench can eliminate some of the problems.
While whitefly transmission of plant viruses can be time consuming and require valuable resources (such as growth rooms) it is essential for the transmission of some viruses for which we have no other means of transmission. It is also a valuable means of screening plants for resistance to viruses, as it uses the same type of transmission that plants in the field will be expected to resist. The use of more artificial means of transmission do not always yield the best results in evaluations of germplasm for virus-resistance.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the Item</td>
			<td>Company</td>
			<td>Catalogue No.</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Whitefly proof screening</td>
			<td>Klayman Meteor Ltd. Petah Tikva 49130, Israel</td>
			<td>---</td>
			<td>BioNet 50 mesh screen Antivirus Insect Screen</td>
		</tr>
		<tr>
			<td>Whitefly proof screening</td>
			<td>Hummert International (www.hummert.com)</td>
			<td>27-4050-1 - 27-4062-1</td>
			<td>BioNet 50 mesh screen Antivirus Insect Screen</td>
		</tr>
		<tr>
			<td>Whitefly Collection Devices</td>
			<td>BioQuip</td>
			<td>1135A</td>
			<td>Can also be made using eye droppers, flexible plastic tubing, plastic vacuum adaptors, and cheesecloth</td>
		</tr>
		<tr>
			<td>Collection vials</td>
			<td>BioQuip</td>
			<td>8909</td>
			<td>Vials used depend on which aspirator is used.</td>
		</tr>
		<tr>
			<td>Yellow plastic cards</td>
			<td>any company</td>
			<td>---</td>
			<td>Any bright yellow plastic card will work, can be purchased in many places.</td>
		</tr>
		<tr>
			<td>Whitefly Cages</td>
			<td>---</td>
			<td>---</td>
			<td>These are not commercially available and must be constructed. 60 cm x 60 cm x 60 cm cages work well for us. Our preference is for those constructed of aluminum window screening, nylon organdy, vinyl plastic, with a structure created using aluminum window frames. Doors are attached using piano hinges and sealed with weather stripping; tops of the cages are vinyl plastic. Cages can also be made of 1) plexiglass glued together on the edges with ventilation provided by screened openings cut on the side pieces; 2) large bags of nylon organdy, organza, or 50 mesh whitefly screening with internal supports provided by PVC pipes and fittings cut to desired lengths and assembled inside the bags, 3) wood frames and nylon organdy, organza, or 50 mesh whitefly screening.</td>
		</tr>
	</tbody>
</table>

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.

These methods for whitefly colony establishment and maintenance, as well as manipulation to transmit plant viruses have been used successfully in a number of studies4,11,12,14 as well as many others not cited. Using these methods with persistently transmitted viruses, we have routinely obtained the desired rates of transmission of 100% for selection for resistance, screening insecticides for their ability to interfere with transmission (and evaluation of resistance inducing compounds)13,14,17,18 (Figure 9). The procedures described here can and have been be adapted to many locations in both public and private research facilities.
<table border="1">
	<tbody>
		<tr>
			<td>Type of Transmission</td>
			<td>Acquisition Access Period (hour)</td>
			<td>Inoculation Access Period (hour)</td>
			<td>Ref.</td>
		</tr>
		<tr>
			<td>Nonpersistent</td>
			<td>1</td>
			<td>2-24</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Semipersistent</td>
			<td>6-24</td>
			<td>8-24</td>
			<td>10, 16</td>
		</tr>
		<tr>
			<td>Persistent</td>
			<td>48-72</td>
			<td>48-72</td>
			<td>3, 6, 8, 9</td>
		</tr>
	</tbody>
</table>
Table 1. Estimated times that can be used to produce high rates of transmission of viruses with different types of relationships with their vector Bemisia tabaci species complex. Estimated times are based on published minimum times required for 80-100% transmission and modified in some cases to account for latent periods and differences among whitefly populations, virus, and host plants.
<img alt="Figure 1" src="/files/ftp_upload/4332/4332fig1.jpg" /> 
Figure 1. An example of a viruliferous whitefly colony. This growth room contains cages of plants infected with a begomovirus and infested with viruliferous whiteflies.
<img alt="Figure 2" src="/files/ftp_upload/4332/4332fig2.jpg" /> 
Figure 2. Left: Immatures stages of the whitefly, Bemisia tabaci MEAM clade sensu De Barro6 approximately 1 week post-emergence and Right: an adult whitefly, an effective vector of many plant viruses.
<img alt="Figure 3" fo:content-width="5.5in" fo:src="/files/ftp_upload/4332/4332fig3highres.jpg" src="/files/ftp_upload/4332/4332fig3.jpg" /> 
Figure 3. Types of collection devices that can be used to collect whiteflies for plant virus transmission.
<img alt="Figure 4" src="/files/ftp_upload/4332/4332fig4.jpg" /> 
Figure 4. Left: Yellow plastic card used to collect whitefly adults showing whiteflies ready for collection. Right: Collecting whiteflies from a yellow plastic card. Whiteflies can be seen on the card as small white/gray spots.
<img alt="Figure 5" src="/files/ftp_upload/4332/4332fig5.jpg" /> 
Figure 5. Whiteflies collected by aspiration (20 in a vial) from the colony ready for either acquisition or inoculation.
<img alt="Figure 6" fo:content-width="5.5in" fo:src="/files/ftp_upload/4332/4332fig6highres.jpg" src="/files/ftp_upload/4332/4332fig6.jpg" /> 
Figure 6. Upper Left: Single plant cages used for either acquisition or inoculation. Upper Right: Larger cages made of pvc pipe and organza. Lower: Aluminum/screen and organdy cages in a greenhouse containing plants intended for a whitefly colony.
<img alt="Figure 7" src="/files/ftp_upload/4332/4332fig7.jpg" /> 
Figure 7. Whiteflies being transferred to a single plant for inoculation (or acquisition).
<img alt="Figure 8" src="/files/ftp_upload/4332/4332fig8.jpg" /> 
Figure 8. Addition of a systemic insecticide drench to terminate the whitefly inoculation access period.
<img alt="Figure 9" src="/files/ftp_upload/4332/4332fig9.jpg" /> 
Figure 9. Field plants under evaluation for resistance to Tomato yellow leaf curl virus. All plants were inoculated as 5 week old seedlings and then transplanted to the field for evaluation. Plants in the foreground are a resistant germplasm line, plants in the background are susceptible germplasm line.

Watch this Scientific Journal Video about Transmitting Plant Viruses Using Whiteflies at JoVE.com

Transmitting Plant Viruses Using Whiteflies

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 ...

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Research

JoVE Journal

Biology

28.7K visualizações.  University of Florida. Melhorar a nossa compreensão e nossa capacidade de gerir muitos dos vírus de plantas a insetos transmitidas requer o uso do vetor. Insecto de transmissão de vírus de plantas é uma interacção tritrófica, e como tal requer a manipulação de insetos, vírus e vegetais. Vectores deve ser criado em grandes números e manipulado de tal forma que para assegurar altas taxas de transmissão para as plantas de teste. As noções básicas de criação e manipulação de mos...

Vídeo: Transmitting Plant Viruses Using Whiteflies

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

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 ...

Immunology and Infection

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce&#39;s disease (PD) of grapevine. Pierce&#8217;s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

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, ...

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.

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. ...

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22)&#160;or&#160;phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus&#160;and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the ...

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.

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 ...

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective ...

Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran insects that have piercing-sucking mouthparts. During viral transmission, the insect saliva bridges the virus-vector-host because the saliva vectors viruses, and the insect proteins, trigger or suppress the immune response of plants from insects into plant hosts. The identification and functional analyses of salivary proteins are becoming a new area of focus in the research field of arbovirus-host interactions. This protocol provides a system to detect proteins in the saliva of leafhoppers using the plant host. The leafhopper vector Nephotettix cincticeps infected with rice dwarf virus (RDV) serves as an example. The vitellogenin and major outer capsid protein P8 of RDV vectored by the saliva of N. cincticeps can be detected simultaneously in the rice plant that N. cincticeps feeds on. This method is applicable for testing the salivary proteins that are transiently retained in the plant host after insect feeding. It is believed that this system of detection will benefit the study of hemipteran-virus-plant or hemipteran-plant interactions.

This protocol demonstrates how to use the plant host to detect salivary proteins of leafhopper and plant viral proteins released by leafhopper vectors.

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran ...