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

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

1. 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.
2. 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.
3. 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

2. Establish Egg Cohorts

1. 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 1^st instar nymphs settle on the same leaf as the egg and the plant adds leaves above the initial oviposition site as it grows.
   1. 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.
2. 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.
   1. 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).
3. 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).
4. 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).
5. 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.
6. 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).
7. Establish each cohort on a single day.
   NOTE: A cohort in a given plot is comprised of a minimum of 50 eggs total.
   1. 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.

3. Establish Nymph Cohorts

1. Use an 8X hand lens to search for newly settled 1^st 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.
2. 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 1^st instar nymphs are called crawlers, which can move several cm during the first few hours after egg hatch. It then "settles" onto a site where it will feed and molt without ever moving again until the adult emerges. These settled 1^st 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.
   1. 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).
3. 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).
4. 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).
5. 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.
6. 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).
7. To ensure that settled 1^st 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.
8. 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 1^st 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.

4. Observation and Recording of Egg Hatch and Mortality

1. After 8-10 d (28-32 °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.
2. 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 °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.

5. Observation and Recording of Nymphal Development and Mortality

1. 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).
   1. 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 °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.
   2. 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 4^th 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)
2. Record development stage (if alive) and cause of death and stage in the notebook initiated at cohort establishment (Table 1).
3. 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.

6. Data Summary and Analyses

1. Consult resources available to help in constructing life tables from the data collected^2,8,11,31. 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.
   1. Estimate real mortality (d_x/l₀) based on the number of insects established at the beginning of the generation.
      Real mortality=(d_x/l₀)
      Where d_x 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 d_x over stages estimates the total mortality rate for the generation (Table 2).
   2. Estimate apparent mortality within a stage (q_x) based on the number of insect alive at the beginning of a specific stage (Table 2). Estimate stage-specific q_x or factor within stage-specific q_x. These rates are additive only within a stage.
   3. Determine marginal mortality using the formula:
      M_B = d_B/(1-d_A)
      Where M_B is the marginal rate of mortality factor B, d_B is the apparent rate of mortality from factor B and d_A is the apparent rate of mortality summed for all mortality factors that can outcompete factor B^13,32 (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 cause^32,34. 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.
      1. 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].
   4. Estimate irreplaceable mortality as [1-e^(-TotalK)] – [1-e^{(-{TotalK – kvalue}_i^})].
      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.
   5. Key factors
      1. 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.
   6. 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 analyses^36,37 can be conducted.