Support was provided by Specialty Crop Block Grants from the Florida Department of Agriculture and Consumer Services Numbers 01856 and 024049. Additional support came from cooperative agreements between the USDA-ARS and the University of Florida Numbers 58-6618-2-096 and 58-6618-4-035. We thank the previous students, postdocs, and collaborators who have contributed in so many ways to our research to understand the factors affecting the population dynamics of flower thrips.

1. Field experiment to determine the effects of UV-reflective mulch, kaolin, and companion plants on flower thrips and their minute pirate bug predator
<ol>
	<li>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.
	<ol>
		<li>Layout blocks of tomatoes or pepper that are each at least 6 m wide and 72 m long.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Kaolin treatment
	<ol>
		<li>Randomly divide each whole plot into equal subplots of kaolin or no kaolin treatments.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Companion plants
	<ol>
		<li>Randomly divide each subplot into equal sub-subplots of companion plants or nor companion plant treatments.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59869/59869fig01.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/59869/59869fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Flower thrips sampling protocol
<ol>
	<li>Prepare 50 mL sample vials before going to the experimental plots.
	<ol>
		<li>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.</li>
		<li>Put exactly 30 mL of 70% alcohol in each 50 mL vial.</li>
		<li>Place the vials into a tray.</li>
		<li>Take the trays to the experimental field site.</li>
	</ol>
	</li>
	<li>Sample the flowers for thrips and minute pirate bugs.
	<ol>
		<li>Randomly assign the tomato or pepper plants to be sampled in each sub-subplot.</li>
		<li>Sample between mid-morning and mid-afternoon.</li>
		<li>Take the samples from the upper half of the plant.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Repeat the sampling of each sub-subplot at least weekly during the flowering period of the crop.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59869/59869fig02.jpg" /> 
Figure 2: Sample removal technique. 
A sample of 10 tomato flowers being collected from a sub-sub plot in the tomato push-pull experiment9. <a href="https://www.jove.com/files/ftp_upload/59869/59869fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Sample processing in the laboratory
<ol>
	<li>Extract the thrips and minute pirate bugs from the flowers in each sample.
	<ol>
		<li>Remove the sample from the refrigerator and tray without disturbing the contents.</li>
		<li>Remove the lid of the vial and carefully extract with a pipette any excess alcohol above the flowers.</li>
		<li>Reseal the vial and shake to dislodge the thrips and minute pirate bugs in the flowers.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Transfer the Petri dish to the platform of a stereoscope with 40x&#8211;150x magnification.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59869/59869fig03.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/59869/59869fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Identify and count the flower thrips in samples.
	<ol>
		<li>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.</li>
		<li>Identify the adult flower thrips species in Florida based on the setae on the pronotum, head and second antennal segment10,11,12.</li>
		<li>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).</li>
		<li>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).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59869/59869fig04.jpg" /> 
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). <a href="https://www.jove.com/files/ftp_upload/59869/59869fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Identify and count the minute pirate bugs in Florida samples.
	<ol>
		<li>Identify and count in each grid the number of adult O. insidiosus and O. pumilio and the number of nymphal Orius species13,15.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Transfer the data from each sample to a spreadsheet that includes the sample date, treatment, and replication.</li>
	<li>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.</li>
	<li>Maintain and manage the datafile with appropriate backup for long-term access.</li>
</ol>

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L., Smith, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Florida+flower+thrips+(suggested+common+name)+Frankliniella+bispinosa+Morgan+(Insecta:+Thysanoptera:+Thripidae).">Florida flower thrips (suggested common name) Frankliniella bispinosa Morgan (Insecta: Thysanoptera: Thripidae).</a> University of Florida, IFAS, EDIS Document EENY639. , (2018).</li><li>Cluever, J. D., Smith, H. A., Funderburk, J. E., Frantz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+flower+thrips+(Frankliniella+occidentalis+[Pergande).">Western flower thrips (Frankliniella occidentalis [Pergande).</a> University of Florida, IFAS, EDIS Document ENY883. , (2018).</li><li>Sprague, D., Funderburk, J., Lucky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flower+thrips,+Frankliniella+tritici+(Fitch).">Flower thrips, Frankliniella tritici (Fitch).</a> University of Florida, IFAS, EDIS Document EENY720. , (2018).</li><li>Herring, J. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identity+of+two+sympatric+species+of+Orius+(Hemiptera:+Heteroptera:+Anthocoridae).">Identity of two sympatric species of Orius (Hemiptera: Heteroptera: Anthocoridae).</a> Journal of Insect Science. 10, 189 (2010).</li><li>Salguero Navas, V. E., Funderburk, J. E., Beshear, R. J., Olson, S. M., Mack, T. 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E., Stavisky, J., Olson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+abundance+and+movement+of+Frankiliniella+thrips+and+Orius+insidiosus+in+field+pepper.">Population abundance and movement of Frankiliniella thrips and Orius insidiosus in field pepper.</a> Agricultural and Forest Entomology. 3, 129-137 (2001).</li><li>Baez, I., Reitz, S., Funderburk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predation+by+Orius+insidiosus+(Heteroptera:+Anthocoridae)+on+life+stages+and+species+of+Frankliniella+flower+thrips+(Thysanoptera:+Thripidae)+in+pepper+flowers.">Predation by Orius insidiosus (Heteroptera: Anthocoridae) on life stages and species of Frankliniella flower thrips (Thysanoptera: Thripidae) in pepper flowers.</a> Environmental Entomology. 33, 662-670 (2004).</li><li>Nakahara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annotated+list+of+the+Frankliniella+species+of+the+world+(Thysanoptera:+Thripidae).">Annotated list of the Frankliniella species of the world (Thysanoptera: Thripidae).</a> Contributions on Entomology, International. 2, 355-389 (1997).</li></ol>

The authors have nothing to disclose.

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 plants6,16, 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 enemies2,18,19. 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.

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 competition3,4. 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 antennae10,11,12.
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 date2,9. 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 thrips2,9.

<table>
	<tbody>
		<tr>
			<td>Alcohol</td>
			<td>Any source</td>
			<td></td>
			<td>70% ethanol or isopropyl</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Fisher Scientific Co.</td>
			<td>06-443-18</td>
			<td>Flat cap and trayed</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific Co.</td>
			<td>08-885</td>
			<td>Medium point</td>
		</tr>
		<tr>
			<td>Kaolin clay</td>
			<td>Novasource</td>
			<td>Surround WP</td>
			<td>95% kaolin</td>
		</tr>
		<tr>
			<td>Pasteur pipet</td>
			<td>Fisher Scientific Co.</td>
			<td>13-678-6A</td>
			<td>5 &#190; inch disposable</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific Co.</td>
			<td>FB0875711A</td>
			<td>With grid</td>
		</tr>
		<tr>
			<td>Probes/seekers</td>
			<td>Fisher Scientific Co.</td>
			<td>08-995</td>
			<td>6 inch bent end</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fisher Scientific Co.</td>
			<td>14-840-00</td>
			<td>Excel international</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>M Series</td>
			<td>40X and greater</td>
		</tr>
		<tr>
			<td>UV-reflective mulch</td>
			<td>Intergro</td>
			<td>Metalized</td>
			<td></td>
		</tr>
	</tbody>
</table>

sampling for estimating frankliniella species flower thrips and orius species predators in field experiments

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.

Data collected in the study by Tyler-Julian et al.9 can be used to demonstrate the separate and combined effects of push factors (i.e., ultraviolet-reflective mulch and kaolin application) and pull factor (i.e., the companion plant Spanish needle, B. alba) on the population dynamics of F. occidentalis adult males and females in tomato flowers (Figure 1A). The agricultural plastic mulch treatments in the experiment were used to form the bed of the raised-bed plastic mulch system that is typical of the production system used to grow high-value vegetables in Florida. The mechanism of the ultraviolet-reflective mulch in pest control is a visual deterrence that disrupts host-finding by the adult thrips. Kaolin application on the tomato plants also reflects enough ultraviolet light to deter the thrips adults. Therefore, a split-split plot randomized complete block design was employed in the experiment to reduce the inter-plot interference on thrips movement resulting from the ultraviolet-reflecting properties of the mulch and kaolin treatments, with mulch treatment (ultraviolet-reflective vs. conventional black mulch) as the whole plot, kaolin treatment (twice weekly kaolin application vs. no kaolin) as the sub-plot, and companion plant treatment (companion plants vs. no companion) as the sub-subplot. Sub-subplot size was six beds by 9 m, with the four inner beds of each sub-subplot consisting of one linear row of tomato with a 45 cm spacing between plants, for a total of 80 plants per sub-subplot. Two rows of Spanish needle were planted into each of the two external beds in the sub-subplots with the companion plant with a 30 cm spacing within and between rows for a total of 128 companion plants per sub-subplot.
Two samples of 10 tomato flowers were collected in each sub-subplot on each of 13 dates in 2011 during the flowering period of the tomato crop, and the number of adult male and female F. occidentalis in each sample were determined (Figure 5). The effects of mulch, kaolin, and companion plant on each gender were analyzed using analysis of variance for a randomized complete block design for a split-split plot treatment arrangement for data across sample date using a mixed model (see Tyler-Julian et al.9 for a complete description of the analysis of variance and results). The main effects of mulch, kaolin, and companion plant were significant for the male western flower thrips (p &#60; 0.01, 0.001, and 0.001, respectively), while the interactive effects of mulch X kaolin, mulch X companion plant, kaolin X companion plant, and mulch X kaolin X companion plant interactions were not significant (p &#62; 0.05). These results showed that each of the main effects reduced the number of adult male F. occidentalis, and that the effects of each tactic were additive when combined with one another.
The main effect of mulch was significant for the female F. occidentalis (p &#60; 0.01), while the main effects of kaolin and companion plants were not significant for the female F. occidentalis (p &#62; 0.05). Therefore, the ultraviolet-reflective mulch reduced the female F. occidentalis in the tomato flowers, but kaolin and the companion plant did not. However, the mulch X kaolin interaction was significant (p &#60; 0.05) showing that the combined effects of ultraviolet-reflective mulch and kaolin reduced the female F. occidentalis more than either tactic alone, while the kaolin applied to tomato on black mulch did not reduce female F. occidentalis numbers. The interactive effects of mulch X companion plant, kaolin X companion plant, and mulch X kaolin X companion plant interactions for female F. occidentalis were not significant (p &#62; 0.05).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59869/59869fig05.jpg" /> 
Figure 5: Example of analysis of data over sample date. 
The mean number per 10 tomato flowers (SEM) of adult male and female F. occidentalis in mulch, kaolin, and companion plant treatments for sample data pooled across 13 dates in 2011 in a push-pull experiment conducted in Gadsden County, Florida. This figure has been modified from Tyler-Julian et al.9
The interaction of mulch X sample date was significant in the experiment in 2011 for male and female F. occidentalis adults (p &#60; 0.01 and 0.001, respectively)9. This revealed that the ultraviolet-reflective mulch reduced flower thrips numbers on some, but not all, sample dates. Therefore, additional analyses were conducted to evaluate the effects of mulch on individual sample dates. The interaction showed that the ultraviolet-reflective mulch was effective in reducing flower thrips numbers early in the season, but there was no significance on individual sample dates during mid- or late-season (Figure 6).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59869/59869fig06.jpg" /> 
Figure 6: Example of population dynamics for whole plot treatment. 
The mean number (+SEM) per 10 tomato flowers (n = 18 samples) of adult male and female F. occidentalis on each 2011 sample date in the whole plot treatment of black and ultraviolet-reflective mulch for data pooled across kaolin and companion plant treatments in the push-pull experiments conducted in Gadsden County, Florida (*indicates significance beyond 95% level of significance according to analysis of variance conducted for individual sample dates; d.f. = 1, 2). This figure has been modified from Tyler-Julian et al.9. <a href="https://www.jove.com/files/ftp_upload/59869/59869fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The interaction of kaolin X sample date was not significant in 2011 for male or female F. occidentalis (p &#62; 0.05)9. As previously shown above, the analyses of data pooled over sample date revealed that kaolin did not significantly affect female F. occidentalis numbers, while male F. occidentalis numbers were significantly reduced. The lack of a significant kaolin X sample date interaction in the analyses for data pooled over sample date suggested that the results for each gender were consistent across sample date (Figure 7).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59869/59869fig07.jpg" /> 
Figure 7: Example of population dynamics for subplot treatment. 
The mean number (+SEM) per 10 tomato flowers (n = 12 samples) of adult male and female F. occidentalis on each 2011 sample date in the subplot treatment of kaolin and no kaolin for data pooled across companion plant treatments in the push-pull experiments conducted in Gadsden County, Florida (*indicates significance beyond 95% level of significance according to analysis of variance conducted for individual sample dates; d.f. = 1, 4). This figure has been modified from Tyler-Julian et al.9. <a href="https://www.jove.com/files/ftp_upload/59869/59869fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The interaction of companion plant X sample date was significant in 2011 for male F. occidentalis (p &#60; 0.05), but not for female F. occidentalis (p &#62; 0.05)9. The analyses conducted to evaluate the effects of companion plant on individual sample dates revealed that companion plants reduced adult F. occidentalis numbers on late season sample dates, but never on early or mid-season sample dates (Figure 8).
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59869/59869fig08.jpg" /> 
Figure 8: Example of population dynamics for sub-subplot treatment. 
The mean number (+SEM) per 10 tomato flowers (n = 6 samples) of adult male and female F. occidentalis on each 2011 sample date in the sub-subplot treatment of companion plant and no companion plant in the push-pull experiments conducted in Gadsden County, Florida (*indicates significance beyond 95% level of significance according to analysis of variance conducted for individual sample dates; d.f. = 1, 8). This&#160;figure has been modified from Tyler-Julian et al.9. <a href="https://www.jove.com/files/ftp_upload/59869/59869fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Data collected from the flowers of the companion plant in the study of Tyler-Julian et al.14 can be used to demonstrate the dynamic relationship between minute pirate bugs and its thrips prey in flowers (Figure 1B). As in the Tyler-Julian et al.9 study, the objectives were to determine the separate and combined effects of push factors (i.e., ultraviolet-reflective mulch and kaolin application) and a pull factor (i.e., the companion plant), on the population dynamics of Frankliniella species adult males and females in crop flowers. In the Tyler-Julian et al.14 study, the predominant flower thrips species was F. bispinosa in the companion plant H. annuus and in the pepper crop (&#62;99% of the total thrips in the flowers). The thrips rapidly colonized the sunflowers and the pepper flowers, and their numbers were greatest soon after flowering began (Figure 9). Populations of thrips declined over time as the numbers of minute pirate bugs increased. The predator-prey ratio illustrated the ability of the predator to suppress the thrips populations with near extinction of the thrips populations occurring at ratios of &#62;1 predator per 40 thrips.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/59869/59869fig09.jpg" /> 
Figure 9: Example of evaluating the benefits of predation. 
Mean number (+SEM) of total thrips (adults and larvae) and total Orius spp. (adults and nymphs) per Helianthus annuus flower head in experiments conducted in 2011 and 2012 in Palm Beach County, Florida (the number of total thrips prey per predator on each date shown in parenthesis). This figure has been adapted from data reported in Tyler-Julian et al.14 with permission from Oxford University Press.

Watch this Scientific Journal Video about Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments at JoVE.com

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

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.

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

The western flower thrips, Frankliniella occidentalis (Pergande), is a polyphagous pest that has been spread worldwide. The extensive use of insecticides ...

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8.8K Views.  University of Florida. 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.

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

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals ...

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

Integrating Remote Sensing with Species Distribution Models; Mapping Tamarisk Invasions Using the Software for Assisted Habitat Modeling (SAHM)

Early detection of invasive plant species is vital for the management of natural resources and protection of ecosystem processes. The use of satellite remote sensing for mapping the distribution of invasive plants is becoming more common, however conventional imaging software and classification methods have been shown to be unreliable. In this study, we test and evaluate the use of five species distribution model techniques fit with satellite remote sensing data to map invasive tamarisk (Tamarix spp.) along the Arkansas River in Southeastern Colorado. The models tested included boosted regression trees (BRT), Random Forest (RF), multivariate adaptive regression splines (MARS), generalized linear model (GLM), and Maxent. These analyses were conducted using a newly developed software package called the Software for Assisted Habitat Modeling (SAHM). All models were trained with 499 presence points, 10,000 pseudo-absence points, and predictor variables acquired from the Landsat 5 Thematic Mapper (TM) sensor over an eight-month period to distinguish tamarisk from native riparian vegetation using detection of phenological differences. From the Landsat scenes, we used individual bands and calculated Normalized Difference Vegetation Index (NDVI), Soil-Adjusted Vegetation Index (SAVI), and tasseled capped transformations. All five models identified current tamarisk distribution on the landscape successfully based on threshold independent and threshold dependent evaluation metrics with independent location data. To account for model specific differences, we produced an ensemble of all five models with map output highlighting areas of agreement and areas of uncertainty. Our results demonstrate the usefulness of species distribution models in analyzing remotely sensed data and the utility of ensemble mapping, and showcase the capability of SAHM in pre-processing and executing multiple complex models.

Integrating Remote Sensing with Species Distribution Models; Mapping Tamarisk Invasions Using the Software for Assisted Habitat Modeling SAHM

We demonstrate the utility of remotely sensed data and the newly developed Software for Assisted Habitat Modeling (SAHM) in predicting invasive species occurrence on the landscape. An ensemble of predictive models produced highly accurate maps of tamarisk (Tamarix spp.) invasion in Southeastern Colorado, USA when assessed with subsequent field validations.

Early detection of invasive plant species is vital for the management of natural resources and protection of ecosystem processes. The use of satellite ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Laboratory and Field Protocol for Estimating Sheet Erosion Rates from Dendrogeomorphology

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to severe environmental impacts. The understanding of sheet erosion is, consequently, a worldwide issue with implications for both environment and economies. However, the knowledge on how erosion evolves in space and time is still limited, as well as its effects on the environment. Below, we explain a new dendrogeomorphological protocol for deriving eroded soil thickness (Ex) by acquiring accurate microtopographic data using both terrestrial laser scanning (TLS) and microtopographic profile gauges. Additionally, standard dendrogeomorphic procedures, dependent on anatomical variations in root rings, are utilized to establish the timing of exposure. Both TLS and microtopographic profile gauges are used to obtain ground surface pro&#64257;les, from which Ex is estimated after the threshold distance (TD) is determined, i.e., the distance between the root and the sediment knickpoint, which allows de&#64257;ning the lowering of the ground surface caused by sheet erosion. For each profile, we measured the height between the topside of the root and a virtual plane tangential to the ground surface. In this way, we intended to avoid small-scale impacts of soil deformation, which may be due to pressures exerted by the root system, or by the arrangement of exposed roots. This may provoke small amounts of soil sedimentation or erosion depending on how they physically affect the surface runoff. We demonstrate that an adequate microtopographic characterization of exposed roots and their associated ground surface is very valuable to obtain accurate erosion rates. This finding could be utilized to develop the best management practices designed to eventually halt or perhaps, at least, lessen soil erosion, so that more sustainable management policies can be put into practice.

Characterizing erosion from dendrogeomorphology has usually focused on accurately finding the starting time of root exposure, by examining macroscopic or cell level changes caused by exposure. Here, we offer a detailed description of different novel techniques to obtain more precise erosion rates from highly accurate microtopographic data.

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to ...

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has become particularly relevant for assessing the anthropogenic-altered global nitrogen cycle and the emission of greenhouse gases (i.e., N2O). Several methods are available for measuring denitrification, but none of them are completely satisfactory. Problems with existing methods include their insufficient sensitivity, and the need to modify the substrate levels or alter the physical configuration of the process using disturbed samples.This work describes a method for estimating sediment denitrification rates that combines coring, acetylene inhibition, and microsensor measurements of the accumulated N2O. The main advantages of this method are a low disturbance of the sediment structure and the collection of a continuous record of N2O accumulation; these enable estimates of reliable denitrification rates with minimum values up to 0.4-1 &#181;mol N2O m-2 h-1. The ability to manipulate key factors is an additional advantage for obtaining experimental insights. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate. The method is appropriate for estimating denitrification rates in any aquatic system with retrievable sediment cores. If the N2O concentration is above the detection limit of the sensor, the acetylene inhibition step can be omitted to estimate the N2O emission instead of denitrification. We show how to estimate both actual and potential denitrification rates by increasing nitrate availability as well as the temperature dependence of the process. We illustrate the procedure using mountain lake sediments and discuss the advantages and weaknesses of the technique compared to other methods. This method can be modified for particular purposes; for instance, it can be combined with 15N tracers to assess nitrification and denitrification or field in situ measurements of denitrification rates.

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

This method estimates sediment denitrification rates in sediment cores using the acetylene inhibition technique and microsensor measurements of the accumulated N2O. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate.

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has ...