We thank D. Ashton, V. Barkley, K. Beimfohr, F. Bojorquez, J. Cantrell, G. Castro, R. Christensen, J. Fearn, C. Jara, D. Meade, G. Owens, L. Rodarte, D. Sieglaff, A. Sonoqui, M. Stefanek, B. Stuart, J. Trejo, A. Slade and E. Yescas for technical assistance. Partial support was provided by USDA-Agricultural Research Service, USDA-National Institute for Food and Agriculture Extension IPM Program and Pest Management Alternatives Special Projects, Cotton Incorporated, Arizona Cotton Growers Association, Cotton Foundations, USDA-CREES, NAPIAP (Western Region), and Western Region IPM Special Projects.

NOTE: The techniques described below are considered partial life tables because they do not explicitly include reproduction or mortality of the adult stages. The term cohort is equivalent to generation because it examines mortality from the egg to the adult stage.
1. Establish Field Sites
<ol>
	<li>Conduct life tables at any time during the growth of the crop once insects are present. The choice of when to initiate studies will depend on the goals and objectives of the research.</li>
	<li>Select two rows of crop near the center of the plot to minimize edge effects from surrounding plots or uncultivated areas. Mark the head of each row with a wire flag or a wooden stake to facilitate easy relocation once the crop gets large.</li>
	<li>Move 3-4 m in from edge of plot parallel to the row. Use one row to establish egg cohorts and the second to establish nymph cohorts</li>
</ol>
2. Establish Egg Cohorts
<ol>
	<li>Use an 8X hand lens to search for newly laid eggs on the underside of leaves near the top of the main stem of the cotton plant (generally the second or third node from the top). 
	NOTE: Life stages of B. tabaci are generally distributed vertically in the plant canopy with eggs near the top of the plant and progressively older nymphal stages below. This results because 1st instar nymphs settle on the same leaf as the egg and the plant adds leaves above the initial oviposition site as it grows.
	<ol>
		<li>Use a higher power 15X lens to observe fresh eggs and verify identification before using the 8x lens to mark the insect. Fresh eggs have a bright white coloration under the lens and stand out from eggs that are older (Figures 1A and 1B). Eggs darken to a tannish color as they mature.</li>
	</ol>
	</li>
	<li>Use a non-toxic, ultra-fine-point black permanent marker to draw a small circle around the egg. Draw the circle small enough to minimize the chance of a female laying another egg within the circle later on.
	<ol>
		<li>Cut a hole or slot in the side of the 8X hand lens with a hacksaw or drill bit so that the pen can be inserted and viewed through the lens when drawing (Figure 2A).</li>
	</ol>
	</li>
	<li>Repeat this process on the same leaf, if possible, to mark other eggs, marking a total of no more than four eggs on a single leaf and no more than one egg per leaf sector. For cotton, the leaf is subdivided into four sectors by three major leaf veins (Figure 3).</li>
	<li>Tie a small lightweight cardboard tag around the petiole of the leaf containing marked egg(s). Number the tag and include notation for the plot or treatment number depending on the experimental design (Figure 2B).</li>
	<li>Tie a 1 m long length of flagging tape around the main stem near the top of the plant. Use a bow-style tie so that the tape can be easily relocated as necessary to keep it visible for repeated visits to this location in the field.</li>
	<li>Record leaf number and positional information on a portable analog or electronic notebook (Table 1). Positional information uses the sectors of the leaf to more finely note location (e.g., 1-1, 1-2, 1-4 denote eggs in sectors 1, 2 and 4 on leaf #1).</li>
	<li>Establish each cohort on a single day. 
	NOTE: A cohort in a given plot is comprised of a minimum of 50 eggs total.
	<ol>
		<li>Use no more than one leaf per plant to better distribute the members of the cohort. 
		NOTE: Depending on insect density, this may comprise anywhere from 13 leaves with 3-4 eggs each or 50 leaves with one egg each. The individual plants are selected to distribute the marked leaves along as much of the row as possible.</li>
	</ol>
	</li>
</ol>
3. Establish Nymph Cohorts
<ol>
	<li>Use an 8X hand lens to search for newly settled 1st instar nymphs on the underside of leaves about 3-5 leaves down from the top of the main stem of the cotton plant. Use a 15X lens to verify identification before marking using the 8X lens.</li>
	<li>Use a non-toxic, ultra-fine-point black permanent marker to draw a small circle around the nymph. Draw the circle small enough to minimize the chance of a crawler settling within the circle later on. 
	NOTE: Newly hatched 1st instar nymphs are called crawlers, which can move several cm during the first few hours after egg hatch. It then &#34;settles&#34; onto a site where it will feed and molt without ever moving again until the adult emerges. These settled 1st instar nymphs (Figure 1C) are distinctive from the crawlers. First, they are immobile and second, they are more 2-dimensional and lay tight and flat on the leaf and have a slightly more translucent amber color.
	<ol>
		<li>Cut a hole or slot in the side of the 8X hand lens with a hacksaw or drill bit so that the pen can be inserted and viewed through the lens when drawing (Figure 2A).</li>
	</ol>
	</li>
	<li>Repeat this process on the same leaf, if possible, to mark other nymphs, marking no more than four nymphs on a single leaf and no more than one nymph per leaf sector. For cotton, the leaf is subdivided into four sectors by three major leaf veins (Figure 3).</li>
	<li>Tie a small lightweight cardboard tag around the petiole of the leaf containing marked nymph. Number the tag and include notation for the plot or treatment number depending on the experimental design (Figure 2B).</li>
	<li>Tie a 1 m long length of flagging tape around the main stem near the top of the plant. Use a bow-style tie so that the tape can be easily relocated as necessary to keep it visible for repeated visits to this location in the field.</li>
	<li>Record leaf number and positional information on a portable analog or electronic notebook (Table 1). Positional information uses the sectors of the leaf to more finely note location (e.g., 1-1, 1-2, 1-4 denote nymphs in sectors 1, 2 and 4 on leaf #1).</li>
	<li>To ensure that settled 1st instar nymphs and not crawlers are marked, go back to each marked leaf and observe the marked insect about 1-2 h after the initial set-up. Remarking of settled nymphs may be necessary.</li>
	<li>Establish each cohort on a single day. 
	NOTE: A cohort in a given plot is comprised of a minimum of 50 nymphs total. No more than one leaf is used per plant to better distribute the members of the cohort. Depending on insect density, this may comprise anywhere from 13 leaves with 3-4 nymphs each or 50 leaves with one nymph each. The individual plants are selected to distribute the marked leaves along as much of the row as possible. Because of the 1st instar crawler stage, the eggs marked in protocol 2 are not the same insects that are then followed as nymphs. Thus, no crawler mortality is measured and the life table is slightly disjointed in time because egg and nymph cohorts are typically established on the same day. Research has shown the crawler mortality is negligible and can be essentially ignored30.</li>
</ol>
4. Observation and Recording of Egg Hatch and Mortality
<ol>
	<li>After 8-10 d (28-32 &#176;C; average Arizona summer conditions) after establishment of egg cohorts, collect leaves containing marked eggs and return to the laboratory for observation under a dissecting microscope. Eggs are too small to clearly evaluate mortality and causes of mortality in the field.</li>
	<li>Determine causes of death for eggs and record in the notebook initiated at cohort establishment (Table 1). 
	NOTE: Death is characterized as dislodgement, predation or inviability. Dislodgement: the egg is missing due to weather events (wind, blowing dust, rain) or chewing predation. Predation: sucking predators leave behind a collapsed chorion (Figure 4K). Hatched eggs can appear collapsed, but there will be a vertical slit in the egg chorion. Use a minuten pin to tease the chorion on the leaf under the microscope to look for this slit. Inviability: eggs fail to hatch after the 8-10 d period and are a dark tan color. Under Arizona summer conditions (28-32 &#176;C) eggs would normally hatch in 5-7 days. This may differ in other regions and adjustments may be necessary in collection time from the field.</li>
</ol>
5. Observation and Recording of Nymphal Development and Mortality
<ol>
	<li>One to two days after cohort establishment, use a 15X lens to assess the development of nymphs and to assign a cause of mortality if dead. Make observations at least three times per week (every other day).
	<ol>
		<li>Use relative size (Figure 1C-G) and time after establishment to assess instar. 
		NOTE: There are four nymphal instars and development is rapid under Arizona summer conditions (28-32 &#176;C) with each of the first three stages lasting around 2 d and the final stage lasting 3-5 d (total nymphal development 2 wk or less). New observers should learn instar sizes by observing insects reared in the laboratory or greenhouse on the host plant of interest. The relative volume in the abdomen of the bacteriosomes (symbiont harboring organs of the whitefly) relative to overall body size is a helpful indicator of nymphal instar (Figure 1C-G). Newly molted nymphs are very flat and translucent. Nymphs ready to molt are more turgid, domed in profile, and opaque in appearance.</li>
		<li>Determine causes of death for nymphs and record in the notebook initiated at cohort establishment (Table 1). 
		NOTE: Death is characterized as dislodgement, parasitism, predation or unknown depending on instar (Figure 4). Dislodgement: nymphs of any stage are missing due to weather events (wind, blowing dust, rain) or chewing predation. Estimate the stage of dislodged nymphs as the average stage of dead and live nymphs on a given observation date. Parasitism: only observable in 4th instar nymphs. The paired yellowish bacterisomes are displaced by the developing parasitoid larva (Figure 4A); the larval stage is sometimes visible (Figure 4C). The pupal stage of the parasitoid is distinctive and genera specific (Figure 4B, 4D). Predation: sucking predators will evacuate the contents of the nymph and leave behind a collapsed cadaver (Figure 4G-4I). Rarely, a chewing predator may leave evidence (Figure 4J). Unknown: death that cannot be attributed to one of the above causes. In humid environments, fungal disease may be an additional cause of death. This category might also include nymphs killed by parasitoid host-feeding. Nymphs that survive emerge as adults leaving a distinctive t-shaped slot in the exuviae (Figure 1H)</li>
	</ol>
	</li>
	<li>Record development stage (if alive) and cause of death and stage in the notebook initiated at cohort establishment (Table 1).</li>
	<li>Once all the nymphs being observed on a single leaf have either died or emerged as an adult whitefly, collect the leaf and return to the laboratory. Use the higher magnification of a dissecting microscope to confirm that the cause of death noted in the field is accurate and make any corrections. 
	NOTE: Not every non-dislodged dead insect will remain on the leaf over the typical two-week observation period and so some verifications will not be possible.</li>
</ol>
6. Data Summary and Analyses
<ol>
	<li>Consult resources available to help in constructing life tables from the data collected2,8,11,31. An example life table is presented as Table 2. 
	NOTE: Robust life table analyses require multiple independent life tables conducted over time and/or different sites. For a multivoltine insect like B. tabaci this could be multiple life tables over the course of a single season and/or multiple seasons and sites.
	<ol>
		<li>Estimate real mortality (dx/l0) based on the number of insects established at the beginning of the generation. 
		Real mortality=(dx/l0) 
		Where dx is the number dying during stage x and l0 is the number of insects at the beginning of the generation. These mortality rates are additive and the sum of dx over stages estimates the total mortality rate for the generation (Table 2).</li>
		<li>Estimate apparent mortality within a stage (qx) based on the number of insect alive at the beginning of a specific stage (Table 2). Estimate stage-specific qx or factor within stage-specific qx. These rates are additive only within a stage.</li>
		<li>Determine marginal mortality using the formula: 
		MB = dB/(1-dA) 
		Where MB is the marginal rate of mortality factor B, dB is the apparent rate of mortality from factor B and dA is the apparent rate of mortality summed for all mortality factors that can outcompete factor B13,32 (Table 3). 
		NOTE: For sessile insects like whiteflies and many other insects, the multiple causes of death within a particular life stage are not sequential. Instead they act contemporaneously and so estimation of marginal rates is required to accurately estimate stage-specific rates of mortality from any one cause32,34. For example, a parasitoid may attack a whitefly nymph. The parasitoid egg might hatch and the larvae may develop in the host. This activity, initially asymptomatic to the observer, does, or would most likely, kill the host insect and should be credited as the cause of death. But in some cases, a predator may attack this same nymph or the nymph may be dislodged from the leaf leading the observer to note the cause of death as predation or dislodgement. Marginal mortality corrects for this.
		<ol>
			<li>Convert marginal stage-specific rates to k-values35 as k = -ln(1-M), where ln is the natural logarithm and M is the marginal mortality rate of interest. k-values are additive and this simplifies further analyses. k-values can be back-converted to proportional mortality rates by 1-e[-k].</li>
		</ol>
		</li>
		<li>Estimate irreplaceable mortality as [1-e(-TotalK)] - [1-e(-{TotalK - kvaluei})]. 
		NOTE: This gives the portion of total generational mortality that would not be realized if a particular mortality factor was removed. For example, how much generational mortality might be lost if predation or parasitism were removed because of an insecticide spray? Irreplaceable mortality estimated in this way assumes that there is no density-dependence in mortality.</li>
		<li>Key factors
		<ol>
			<li>Use a simple graphical analysis to plot the k-value for any one stage, or any one mortality factor (or one mortality factor within one stage) against the Total-K value for the entire generation (Total K = sum of all individual k-values). 
			NOTE: The mortality factor that most closely mimics the pattern of Total-K the best is the key factor, the factor that contribute the most to changes in generational mortality35. A more quantitative method regresses individual k-values on Total-K and identifies the key factor as the one with the greatest slope value3.</li>
		</ol>
		</li>
		<li>Test density dependence by regressing k-values for factors of interest on the natural log of insect population density measured independently (e.g.13). A significant positive slope suggests direct density dependence and a negative slope inverse dependence. 
		NOTE: With additional life table information on adult survival and reproduction, many additional parameters (e.g., generation time, net reproductive rate, instantaneous rates of increase, life expectancy at a given stage, etc.) and analyses (matrix models and elasticity analyses36,37 can be conducted.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Typically, the development of life tables for multivoltine insects with broadly overlapping generations are constrained to a vertical approach where a population is sampled repeatedly over time and various graphical and mathematical techniques are then used to estimate recruitment to the various stages and infer rates of mortality from changing densities of the various life stages2. The strength of the approach here is that it navigates this limitation by isolating a group of immobile equal-aged i...

Life tables are a common tool with a long history in ecology1,2. Life tables are essentially a schedule of the births and deaths in a population over time and such data can be used to quantify a number of parameters important to understanding and predicting population dynamics. Life tables may also provide information on causes of death that are important to understanding trophic interactions and in developing control strategies for managing pests in agricultural and natural systems. Numerous field-based life tables have been constructed for insects3,4,5, and analyses have provided important insights into the dynamics, regulation and prediction of insect populations in many managed and natural systems6,7,8,9,10,11,12,13,14. The term life table is also often used to describe laboratory based studies that largely examine schedules of births and deaths but under artificial conditions that do not expose the insect to natural mortality forces and realistic environmental variables. Generally, the goal of laboratory studies is to estimate the comparative biotic potential of a species. The focus of the methods described here is for field based investigations that define realized potential relative to the environment.
Life tables can be characterized as horizontal, in which a real cohort of equal aged individuals are followed from the beginning of their lives until death, or vertical, where frequent samples are taken through time of a population with an assumed stable age structure and then vital rates are inferred from mathematically constructed cohorts2,15. The type of life table that can be deployed depends on the nature of the insect. Horizontal life tables can often be developed for univoltine (one generation per year) insects, while such an approach can be very challenging for a multivoltine insect with multiple and widely overlapping generations each year. A host of analytical methods have been proposed and used to develop vertical life tables for insect populations (see Southwood2 for examples). The methodology demonstrated here allows for the development of cohort-based, horizontal life tables in the field for multivoltine insects with specific life history characteristics, notably, the presence of sessile life stages. The method is demonstrated for a key pest in cotton as a model system.
The whitefly, Bemisia tabaci biotype B (= Bemisia argentifolii, Middle East-Asia Minor 116) is a global pest of agriculture that negatively impacts yield and quality in many agronomic and horticultural crops, including protected agricultural systems in temperate regions17. Impacts occur due to phloem feeding that disrupts nutrient flow, disorders of unknown etiology caused by nymphal feeding, transmission of numerous plant viruses and crop quality effects due to the deposition of honeydew18,19. The insect has a broad host range and is multivoltine, having as many as 12-13 generations per year depending on region and available food resources20. Management challenges also are exacerbated by its high reproductive potential, its ability to disperse and migrate within and between agricultural systems, its lack of a quiescent stage (diapause or estivation) and its disposition to rapidly develop resistance to insecticides used for suppression21,22.
Considerable progress has been made in developing integrated pest management (IPM) strategies to effectively and economically manage populations of this pest in affected crops23,24,25. These management systems were predicated on a sound fundamental understanding of the population dynamics of B. tabaci and life tables have been a key technique that have enabled this understanding. In Arizona, life tables have allowed the estimation and identification of important mortality forces for B. tabaci in multiple crop systems13,26, have enabled the measurement of mortality dynamics relative to management strategies including non-target effects of insecticides14, have provided a means of estimating potential functional non-target effects of transgenic cotton producing insecticidal proteins27, have supported rigorous assessment of a classical biological control program28 (Naranjo, unpublished data) and helped to explore the comparative effects of top-down and bottom-up effects on pest dynamics29. All of these applications have deployed the methodology described here. The approach could be useful for the study of insect population ecology in a number of natural and managed systems.

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methodology for developing life tables for sessile insects in the field using the whitefly, bemisia tabaci, in cotton as a model system

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

An example cohort is presented in Table 2 to show a typical presentation and calculation of life table results. The most useful data is captured in the marginal mortality rates for each factor within each stage. By converting these rates to k-values (protocol section 6), stage-specific mortality over all factors and factor-specific mortality over all stages can be easily estimated, as can total generational mortality. This also facilitates irreplaceable mortality, key-fac...

Watch this Scientific Journal Video about Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System at JoVE.com

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

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11.8K Views.  USDA-ARS, Arid-Land Agricultural Research Center. Leven tabellen toestaan van kwantificering van de bronnen en de tarieven van sterfte in de insectenbevolking en bijdragen aan het begrijpen, voorspellen en manipuleren van de populatiedynamiek in agro-ecosystemen. Methoden voor het uitvoeren en analyseren van de cohort gebaseerde leven tabellen in het veld voor een insect met sessiele onvolwassen levensstadia worden gepresenteerd.

Video: Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC50) and lethal dose (LD50) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC50 values for insecticide evaluations.

A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or ...

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 ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Biology

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, ...

Immunology and Infection

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 ...

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. ...

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 ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Assessment of Phytochemical Effects on Helicoverpa armigera Through Feeding Assay

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

Author Spotlight: Development and Challenges of Feeding Assays for Insect Pest Management 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

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