Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments

Summary

Presented here is a protocol to determine the number of thrips and minute pirate bug predators in crops over multiple dates in field experiments. Also illustrated is how to determine the efficacy of management tactics against thrips and evaluate the benefits of predation by minute pirate bugs.

Abstract

The western flower thrips, Frankliniella occidentalis (Pergande), is a polyphagous pest that has been spread worldwide. The extensive use of insecticides in attempts to control its populations eliminates natural enemies and competitor flower thrips species, thereby increasing its populations. An unsustainable situation develops with concomitant resistant pest populations, secondary pest outbreaks, and environmental degradation. Integrated pest management utilizes knowledge of pest and natural enemy relationships to implement tactics that are environmentally friendly and sustainable. Minute pirate bugs are the most important worldwide predators of thrips. They can suppress and ultimately control Frankliniella species flower thrips. Flower samples taken at least weekly are needed to understand predator-prey dynamics. Shown here is the sampling of the flowers of fruiting vegetables and companion plants to estimate the densities of individual thrips and minute pirate bug species. Representative data illustrates how the protocol is used to determine the efficacy of management tactics over time and how to evaluate the benefits of predation by minute pirate bugs. The sampling protocol is similarly adaptable to sampling thrips and minute pirate bugs in other plant species hosts.

Introduction

The western flower thrips, Frankliniella occidentalis (Pergande), was one of the first great pests that was spread worldwide as a result of globalism and the international trade of agricultural products. Economic damage results directly from feeding and oviposition and indirectly through transmission of plant pathogenic viruses. Invasive populations were already largely resistant to most classes of insecticides, and the attempts to control populations with insecticides has only increased damage by eliminating important natural enemies and competitor species. This control approach has destabilized management programs and has resulted in resistant pest populations, secondary pest outbreaks, and environmental degradation¹.

Integrated pest management programs have been developed from knowledge of pest and natural enemy relationships and the effects of management tactics on these relationships. The population characteristics of rapid colonization and growth have been long believed to outstrip the capacities of natural enemies to regulate the opportunistic western flower thrips; that is, until it was shown that predation from natural populations of Orius insidiosus (Say) not only resulted in suppression of western flower thrips populations but also a decline of populations toward extinction². Furthermore, the western flower thrips is mostly flower-inhabiting, in which it competes for pollen and other flower resources with native polyphagous flower thrips.

In most of the eastern United States, the main native competitor is Frankliniella tritici (Fitch), while in southern Florida the main competitor species is Frankliniella bispinosa (Morgan)³. The western flower thrips suffers strong biotic resistance in Florida from the native species of predators and competitor flower thrips species; however, it is the dominant species in habitats disturbed by insecticides and other tactics that exclude competitor thrips and natural enemies. Therefore, a core component of successful integrated pest management programs for fruiting vegetables is increased predation and competition^3,4. These programs have been developed from knowledge of predator-prey dynamics and the effectiveness of various tactics to manage thrips and increase biotic resistance. Here, the methodology used to estimate the densities of individual thrips and minute pirate bug species in the flowers of fruiting vegetables and companion plants in Florida are shown. The data are used to determine the efficacy of management tactics and evaluate the benefits of predation by minute pirate bugs.

Designing the flower thrips sampling protocol: background information

When the western flower thrips emerged as a major pest in the 1980s⁵, it was necessary to develop procedures to accurately, efficiently, and precisely determine the number of individual thrips species in field studies. The procedures described here have been developed from knowledge gained in numerous studies conducted to understand the biology and management of flower thrips. Examples of these studies include work by Funderburk et al.², Hansen et al.⁶, Salguero Navas et al.⁷, Sutherland et al.⁸, and Tyler-Julian et al.⁹. The concentrations of Frankliniella species and minute pirate bugs in flowers is behaviorally based and not an artifact of insecticide applications or sampling⁶. Estimates of populations in flowers over other plant parts usually is sufficient for understanding the local dynamics of predator and prey on a plant host and evaluating the benefits of biological control programs based on the predator to prey ratios. However, the methodology developed for flowers can be adapted to the sampling of other plant parts. The usual sample unit is one or more flowers. The number of samples needed to achieve the desired level of precision is a function of population density and number of flowers in the sample unit.

Species of Frankliniella tend to be an aggregated distribution in flowers, and populations usually are concentrated in the flowers of the upper plant canopy⁷. For most studies, flowers are randomly selected from the upper half of the plant. Relative techniques to remove thrips from flowers, including liquid washing, mechanical dislodgement, or desiccation, are inaccurate and imprecise⁸. For this reason, a direct counting, absolute estimate technique is used. Thrips are small organisms about 2 mm in Iength, and microscopy usually is necessary to accurately determine the species. The flowers constituting a sample unit are placed in a vial of 70% alcohol. Once the samples are collected, the vials from each plot are returned to the laboratory for extraction of the thrips and minute pirate bugs and accurate determination of the sex, species, and stage of each. Experiments consist of replicated field plots that are used to evaluate the efficacy of treatments to suppress thrips and the benefits of predation by minute pirate bugs. Flower samples are taken at least weekly during the flowering period of the plant host. Randomized complete block experimental designs are useful in removing from the experimental error differences in thrips and minute pirate bug densities between blocks. Sub-plot treatment arrangements are useful to reduce inter-plot effects of management tactics that affect thrips movement⁹.

Flower sample processing and analysis: background information

Prior to the 1990s, keys to species of thrips were developed for use by taxonomic specialists, who placed thrips for identification onto microscope slides using one of several mounting media. Researchers studying thrips biology and management were not taxonomic specialists, and there was no involvement by taxonomic specialists in the studies. Typically, the thrips in samples from these studies were lumped into the genus, family, suborder, or order levels of classification. After the spread of the western flower thrips, there was 1) rapid proliferation of research concerning thrips biology and management and 2) recognition by researchers for the need to identify thrips species and develop an efficient system for processing samples.

In studies in the mid-1990s involving thrips population biology, adult thrips from the samples were placed onto microscope slides and identified to species by taxonomic specialist R. J. Beshear (e.g., Salguero Navas et al.⁷). The larvae were only identified to genus due to a lack of larval identification keys available at that time. The slide mounting was costly and laborious, and a more efficient system was developed². In subsequent studies, the thrips in the samples were extracted from the flowers in a Petri dish containing 70% alcohol, and the males and females in the Petri dish were identified to species under stereoscopy. Most of our research involves species of Frankliniella. The adults of these species were separated to species under the stereoscope using differences in their chaetotaxy on the dorsal surface of the pronotum, head, and antennae^10,11,12.

Additional expertise in thrips taxonomy has been acquired to recognize and identify other thrips genera and species in the samples. There are numerous Orius species worldwide that are important predators of thrips. Two species, O. insidiosus and O. pumilio (Champion), are sympatric throughout much of Florida¹³. The adults of these species are separated by color characteristics of the basal antennal segment, femora of the hind leg, and cuneus on the wing. Thrips species and genders differ in their biology and behavior; therefore, data for each are typically separately analyzed. Because the thrips populations in flowers have an aggregated pattern of distribution, the data needs transformation to stabilize variances between treatments. Treatment means are compared using analysis of variance as appropriate for the experimental design, and the data is analyzed for each individual date and/or for data pooled over date^2,9. The analysis of effects on individual dates is important when treatment differences vary over date. The ratio of the total thrips (adults and larvae) per minute pirate bug (adults and nymphs) is used to evaluate the effectiveness of biological control with minute pirate bugs in Florida field studies suppressing thrips populations at a ratio of about one predator for every 180 thrips^2,9.

Protocol

1. Field experiment to determine the effects of UV-reflective mulch, kaolin, and companion plants on flower thrips and their minute pirate bug predator

1. Establish a field experiment with a split-split-plot treatment arrangement in a randomized complete block experimental design with mulch type as whole plot treatments, kaolin and no kaolin as subplot treatments, and companion plants and no companion plants as sub-subplot treatments (Figure 1A,B)^9,14.
   1. Layout blocks of tomatoes or pepper that are each at least 6 m wide and 72 m long.
   2. Randomly lay in each block whole plots of black and UV-reflective mulch, with each whole plot consisting of six raised mulch beds at least 36 m long.
   3. Plant one linear row of tomatoes every 45 cm or two linear rows of peppers every 30 cm into the four inner beds of each whole plot.
2. Kaolin treatment
   1. Randomly divide each whole plot into equal subplots of kaolin or no kaolin treatments.
   2. Apply kaolin once or twice weekly at the rate of 7.0 kg/ha to the tomato or pepper plants in the subplots assigned to receive kaolin treatment.
3. Companion plants
   1. Randomly divide each subplot into equal sub-subplots of companion plants or nor companion plant treatments.
   2. Plant two linear rows of Bidens alba (L.) every 30 cm or one linear row of Helianthus annuus L. every 30 cm into the two outer beds of each sub-subplot treatment with companion plants.

Figure 1: Example experimental field study.
(A) Randomized complete block design to evaluate the separate and interactive effects of companion plants, mulch, and kaolin effects on flower thrips and minute pirate bugs. (B) Bidens alba (L.) evaluated as a companion plant species with tomato as the crop⁹. Helianthus annuus L. evaluated as a companion plant species with pepper as the crop¹⁴. Please click here to view a larger version of this figure.

2. Flower thrips sampling protocol

1. Prepare 50 mL sample vials before going to the experimental plots.
   1. Place a label with the mulch, kaolin, and companion plant treatment, the block number, and the sample date on the outside and inside of each vial.
   2. Put exactly 30 mL of 70% alcohol in each 50 mL vial.
   3. Place the vials into a tray.
   4. Take the trays to the experimental field site.
2. Sample the flowers for thrips and minute pirate bugs.
   1. Randomly assign the tomato or pepper plants to be sampled in each sub-subplot.
   2. Sample between mid-morning and mid-afternoon.
   3. Take the samples from the upper half of the plant.
   4. Remove the vial lid. Using a sharp-edged razor or scissors, carefully remove the flower from the plant. Quickly place the flower into the appropriate pre-labeled vial. Push the flower into the alcohol of the vial (Figure 2). Replace the lid.
   5. Collect 10 flowers per sample. Make sure each vial is tightly sealed, then shake each vial to ensure that the flowers are within the alcohol.
3. Return the trays with samples to the laboratory for storage. To ensure samples do not deteriorate prior to processing, keep samples cool and dry. Refrigerate, if possible, especially for samples that are not processed quickly.
4. Repeat the sampling of each sub-subplot at least weekly during the flowering period of the crop.

Figure 2: Sample removal technique.
A sample of 10 tomato flowers being collected from a sub-sub plot in the tomato push-pull experiment⁹. Please click here to view a larger version of this figure.

3. Sample processing in the laboratory

1. Extract the thrips and minute pirate bugs from the flowers in each sample.
   1. Remove the sample from the refrigerator and tray without disturbing the contents.
   2. Remove the lid of the vial and carefully extract with a pipette any excess alcohol above the flowers.
   3. Reseal the vial and shake to dislodge the thrips and minute pirate bugs in the flowers.
   4. Open the vial and pour the contents into a Petri dish. Rinse the inside of the vial with 70% alcohol and pour the contents into the Petri dish. Make sure all thrips and minute pirate bugs in the vial are flushed into the Petri dish.
   5. Dissect each flower with forceps and rinse with 70% alcohol to ensure that all the thrips and minute pirate bugs have been dislodged. Remove and discard the flower parts from the Petri dish (Figure 3).
   6. Transfer the Petri dish to the platform of a stereoscope with 40x–150x magnification.

Figure 3: Extracting thrips and minute pirate bugs from flowers.
A sample of 10 tomato flowers poured into a Petri dish for processing to determine the number of thrips and minute pirate bugs. Please click here to view a larger version of this figure.

2. Identify and count the flower thrips in samples.
   1. Identify and count in each grid the number of adult males and females of each flower thrips species and the number of Frankliniella species larvae.
   2. Identify the adult flower thrips species in Florida based on the setae on the pronotum, head and second antennal segment^10,11,12.
   3. Separate the adults of F. bispinosa from the adults of F. tritici and F. occidentalis by the extra stoutness of the two setae on the anterior dorsal margin of the second antennal segment (Figure 4).
   4. Separate the adult F. occidentalis from those of F. bispinosa and F. tritici by the near equal lengths of the anterior marginal and anterior angular major setae on the pronotum and by the longer fourth postocular setae on the head (Figure 4).

Figure 4: Examples of morphological characters to identify thrips.
(A,B,C) F occidentalis: head (A), arrows indicate postocular setae pair IV; pronotum (B), arrows indicate pair of long anteromarginal setae; distal dorsal setae of antenna segment II (B). (D,E,F,G) . F. bispinosa: head (D); pronotum (E); distal dorsal setae of antenna segment II (F,G), arrow indicates stout setae (F), lateral view of stout setae (G). (H,I,J). F. tritici: head (H); pronotum (I); distal dorsal setae of antenna segment II (J). Please click here to view a larger version of this figure.

3. Identify and count the minute pirate bugs in Florida samples.
   1. Identify and count in each grid the number of adult O. insidiosus and O. pumilio and the number of nymphal Orius species^13,15.
   2. Identify the adult O. insidiosus by the brown basal antennal segments, by the femora that have dark markings, and by the cuneus that is dark brown.
   3. Identify the adult O. pumilio by the yellow basal antennal segments, the yellow or straw-colored femora, and by the cuneus with pale straw or light-brown color.
4. Add the numbers from each grid to determine the total number of adult males and females of each flower thrips species, the number of Frankliniella species larvae, the number of adult minute pirate bug of each species, and the number of minute pirate bug nymphs in the sample.
5. Select representative vouchers of flower thrips and minute pirate bug adults from the samples. Label by date, plant host, location, and collector. Curate for long-term preservation.
6. Transfer the data from each sample to a spreadsheet that includes the sample date, treatment, and replication.
7. Create a data file that contains the data from each sample. Include the experimental location, experimental design, and the amounts and dates of each cultural practice used to establish and maintain the experiment.
8. Maintain and manage the datafile with appropriate backup for long-term access.

Discussion

Sampling protocols with desired levels of precision to estimate population densities of flower thrips were developed for Florida crops over more than three decades of field research. Studies were conducted to understand important aspects of flower thrips biology that affect population estimates. For example, studies were conducted to understand the effects on estimates of time of day when sampling¹⁶, sample location within the field¹⁶, sample location on individual plants^6,16, patterns of aggregation in flowers⁷, and flower color¹⁷. These factors were found to influence population estimates; so, the decisions of where, when, and how are critically important when designing the sampling protocol in future research studies.

Minute pirate bug adults and nymphs are highly anthophilous, and the predator aggregates with its prey in a density-dependent manner by preferring the same flowers also preferred by the thrips¹⁷. They also exploit cues from prey or from plants damaged by prey in locating thrips. The adults move rapidly between flowers, a behavior that enhances their ability to track local populations of thrips prey in space and time¹⁸. Therefore, sampling protocols developed for estimating populations of thrips should be used in future studies when estimating populations of minute pirate bugs. Minute pirate bugs are efficient predators of the adults and larvae of different Frankliniella species flower thrips¹⁹. The number of the predator relative to the number of total thrips prey provides the best estimate of the ability of the minute pirate bugs to suppress and control mixed populations of thrips in the flowers. This should be considered when analyzing the data in future studies.

The Frankliniella species adults rapidly colonize host crops once flowering begins, and rapid population growth follows in the absence of mortality from natural enemies^2,18,19. On a good plant host for both predator and thrips prey such as sunflower, numbers of thrips are greatest soon after flower initiation, followed by declines in populations as minute pirate bugs increase (Figure 9). Populations of minute pirate bugs remain even though thrips are nearly extinct. To fully understand this predator-prey dynamic relationship, it is necessary to sample frequently throughout the flowering period of the crop. The same is true when investigating the efficacy of other types of tactics, as they may be efficacious on some dates and not others. Once or twice weekly sampling over the entire period of flowering was employed to evaluate the effects of multiple tactics in the push-pull system under investigation in Tyler-Julian et al.^9,14.

Frankliniella is the second largest genus in the family Thripidae, and there is a large amount of literature describing the adult life stages²⁰. A complex of species inhabits flowers that is specific to different plant host species and geographic locations. Therefore, expert identification of slide-prepared specimens from a subset of the initial samplings is critical. Then, in any given plant host and geographic location, taxonomic characters unique to the adults of each species can be chosen so that the species can be determined in future studies without going to the laborious and costly procedure of placing each onto microscope slides for viewing under a compound microscope. They simply can be viewed and identified under a stereoscope. (In some unusual situations, the morphological characters separating two species are so similar that they cannot be separated under the stereoscope.) Methods described here for the flower thrips species common in most crops in Florida should be adapted and used at other geographic locations when processing the large numbers of samples needed in field studies to determine the efficacy of management tactics and to evaluate the benefits of predation by minute pirate bugs.

Materials

Name	Company	Catalog Number	Comments
Alcohol	Any source		70% ethanol or isopropyl
Centrifuge tube	Fisher Scientific Co.	06-443-18	Flat cap and trayed
Forceps	Fisher Scientific Co.	08-885	Medium point
Kaolin clay	Novasource	Surround WP	95% kaolin
Pasteur pipet	Fisher Scientific Co.	13-678-6A	5 ¾ inch disposable
Petri dish	Fisher Scientific Co.	FB0875711A	With grid
Probes/seekers	Fisher Scientific Co.	08-995	6 inch bent end
Scalpel	Fisher Scientific Co.	14-840-00	Excel international
Stereomicroscope	Leica Microsystems	M Series	40X and greater
UV-reflective mulch	Intergro	Metalized