We thank Molly Stedfast for dilution and pesticide formulation assistance. We also thank Troy Anderson for probit analysis guidance and Zachary Adelman for assistance with experimental design. This research was partly supported by an Entomological Foundation award and by Virginia Pest Management scholarship funds.

NOTE: This protocol includes two insecticide assays for separately treating bed bug eggs and first instars. Both protocols were conducted using the same insecticides, however, the protocols had to be adapted to ensure insecticide exposure and to easily manipulate the specimens.
1. Egg Dipping Insecticide Assay
<ol>
	<li>Pull mated, well-fed adult female bed bugs from a colony and place the females (5-10) into a Petri dish with filter paper. Have at least ten replicates to ensure that there will be enough eggs for the insecticide assay. Check to see if the females have laid eggs every day.
	<ol>
		<li>Once they lay eggs, take the females out of the Petri dish (6 cm &#215; 5 cm) and put them into new Petri dishes with a new filter paper daily and record the date on the old and new Petri dishes.</li>
		<li>Allow the eggs to age 4 to 5 days before starting the assay. Susceptibility to insecticides changes during embryonic development, so it is important to control for egg age. Hold all life stages at 12:12 L:D photoperiod, 60% RH, and 27 &#176;C.</li>
	</ol>
	</li>
	<li>Once the eggs have aged appropriately, remove the bed bug eggs gently from filter papers using soft-tip forceps. Gently scrape the bottom of the egg attached to the filter paper using the forceps and then gently grab without applying much pressure on the egg to limit throwing bed bug eggs.</li>
	<li>Place the removed bed bug eggs into their respective groups inside of plastic Petri dishes. Limit static electricity from flinging bed bug eggs by rubbing the bottom of each Petri dish on the outside with a fabric softener sheet. Treat groups of bed bug eggs (5-10 eggs per group) all at once. Consider one group a replicate and use at least five (or more) replicates for this assay.</li>
	<li>Prepare serial dilutions of insecticides to get a range of 5 different concentrations (0.21-21 &#181;L/mL for imidacloprid (0.1%)/&#946;-cyfluthrin (0.5%) and a control solution with solvent (tap water) only. Make 20 mL of each concentration to have enough volume for the dipping assay.</li>
	<li>Cut centrifuge tubes (50 mL) at the 35 mL line medially using a rotary tool with a cut-off wheel accessory and then cut a hole into the cap of the centrifuge tube (diameter = 2.54 cm) using the same rotary tool with an aluminum oxide grinding stone accessory. Leave 5 cm on the edge of the cap when cutting, so that the cap will securely screw into the tube (Figure 1).</li>
	<li>Cut fine mesh into squares large enough to fit into the cap (~5 cm2). Screw the mesh tightly, eliminating any bumps, into the centrifuge tubes using the centrifuge tube cap.</li>
	<li>Place a group of bed bug eggs (one replicate) using a paint brush onto the mesh inside of the centrifuge tube. Dip the group of bed bugs inside of the cut centrifuge tube into an insecticide concentration for 5 seconds.</li>
	<li>Remove the centrifuge tube out of the insecticide solution. Dry the mesh with the eggs inside by placing the centrifuge tube directly onto a laboratory tissue.</li>
	<li>Remove the group of eggs out of the centrifuge tube using a small, fine-bristle paint brush and place them into a clean Petri Dish with a clean filter paper.</li>
	<li>Record egg mortality (failure to hatch) for 14 days to ensure all eggs have had sufficient time to hatch.</li>
	<li>Correct for mortality in the control treatment using Abbott&#39;s formula (% test mortality -% control mortality/ 100 - control mortality x 100).</li>
	<li>Insert recorded mortality with the corresponding concentrations into the analysis software15 to calculate LC50 values.</li>
</ol>
2. First Instar Insecticide Assay
<ol>
	<li>Separate a group of well-fed bed bugs (5 adult males and 5 adult females), preferably fed 1 day prior, and place them into a Petri dish (6 cm &#215; 5 cm) with filter paper provided for mating and egg laying. Allow the adult bed bugs to mate and lay eggs for approximately 7-9 days. For feeding, use an artificial feeding system that circulates hot water in glass tubes surrounding blood16. For this study, use defibronated rabbit blood.</li>
	<li>Remove the adults from the Petri dishes (6 cm &#215; 5 cm) and allow the eggs to hatch into first instars within the Petri dishes. It takes approximately 7-9 days for bed bug eggs to hatch at room temperature.</li>
	<li>Collect the unfed first instars of the same age using a paint brush. Age can be determined by removing the first instars daily and recording the day they hatched. Place them into new, clean Petri dishes until the insecticide assay. First instar nymphs are not very desiccant resistant, so the insecticide assay should be completed as soon as possible.</li>
	<li>For the assay, prepare serial dilutions of imidacloprid/&#946;-cyfluthrin to get a range of 5 different concentrations and a control solution with solvent only. Formulated products labeled for bed bug control in the United States were used for these studies, therefore the solvent was water (according to the label).</li>
	<li>Pipette 150 &#181;L aliquots of each insecticide concentration and water for the control group onto individual filter papers (#1; 4.2 cm diameter). Distribute the insecticide over the entire filter paper using the pipetter by applying droplets around the entire edge and center of the filter paper.</li>
	<li>Place the treated filter papers individually on top of individual hardboard panels (7 cm2).</li>
	<li>Release groups of first instars (5 to 10 per group) onto the individual filter papers while still wet with insecticide and cover the first instars with an inverted bottom of a Petri dish.</li>
	<li>Place a weight on top of the Petri dish to prevent the first instar bed bugs from escaping the treated surface.</li>
	<li>Record mortality of first instar bed bugs after 24 h exposure to all insecticide concentrations.</li>
	<li>Correct for mortality in the control treatment using Abbott&#39;s formula (% test mortality -% control mortality/ 100 - control mortality x 100).</li>
	<li>Insert recorded mortality and concentrations into the analysis software15 to calculate LC50 values.</li>
</ol>

<ol>
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Entomol. 23, 163-166 (2009).</li><li>Leverkus, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bullous+allergic+hypersensitivity+to+bed+bug+bites+mediated+by+IgE+against+salivary+nitrophorin.">Bullous allergic hypersensitivity to bed bug bites mediated by IgE against salivary nitrophorin.</a> J. invest. entomol. 126, 91-96 (2006).</li><li>Fletcher, C. L., Ardern-Jones, M. R., Hay, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+bullous+eruption+due+to+multiple+bed+bug+bites.">Widespread bullous eruption due to multiple bed bug bites.</a> Clin. Exp. Dermatol. 27, 74-75 (2002).</li><li>Pritchard, M. J., Hwang, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+anemia+from+bedbugs.">Severe anemia from bedbugs.</a> Can. Med. Assoc. J. 181, 287-288 (2009).</li><li>Goddard, J., de Shazo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychological+effects+of+bed+bug+attacks+(Cimex+lectularius+L).">Psychological effects of bed bug attacks (Cimex lectularius L).</a> Am. J. Med. 125, 101-103 (2012).</li><li>Harlan, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bed+bugs+101:+the+basics+of+Cimex+lectularius.">Bed bugs 101: the basics of Cimex lectularius.</a> Am. Entomol. 52, 99-101 (2006).</li><li>Pinto, L. J., Cooper, R., Kraft, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bed Bug Handbook: The Complete Guide to Bed Bugs and Their Control. , (2007).</li><li>Toloza, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+patterns+of+insecticide+resistance+in+eggs+and+first+instars+of+Triatoma+infestans+(Hemiptera:+Reduviidae)+from+Argentina+and+Bolivia.">Differential patterns of insecticide resistance in eggs and first instars of Triatoma infestans (Hemiptera: Reduviidae) from Argentina and Bolivia.</a> J. Med. Entomol. 45, 421-426 (2008).</li><li>Cueto, G. M., Zerba, E. N., Picollo, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+pyrethroid+resistance+in+eggs+of+Pediculus+humanus+capitis+(Phthiraptera:+Pediculidae)+from+Argentina.">Evidence of pyrethroid resistance in eggs of Pediculus humanus capitis (Phthiraptera: Pediculidae) from Argentina.</a> J. Med. Entomol. 45, 693-697 (2008).</li><li>Ho, S. H., Goh, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deltamethrin+as+a+potential+ovicidal+pyrethroid+against+Plutella+xylostella+L.">Deltamethrin as a potential ovicidal pyrethroid against Plutella xylostella L.</a> Toxicol. let. 22, 161-164 (1984).</li><li>Bell, C. H., Hole, B. D., Evans, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occurrence+of+resistance+to+phosphine+in+adult+and+egg+stages+of+strains+of+Rhyzopertha+dominica+(F.)(Coleoptera:+Bostrichidae).">The occurrence of resistance to phosphine in adult and egg stages of strains of Rhyzopertha dominica (F.)(Coleoptera: Bostrichidae).</a> J. Stored Prod. Res. 13, 91-94 (1977).</li><li>Campbell, B. E., Miller, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insecticide+Resistance+in+Eggs+and+First+Instars+of+the+Bed+Bug,+Cimex+lectularius+(Hemiptera:+Cimicidae).">Insecticide Resistance in Eggs and First Instars of the Bed Bug, Cimex lectularius (Hemiptera: Cimicidae).</a> Insects. 6, 122-132 (2015).</li><li>Price, L. A., Mills, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+toxicity+of+phosphine+to+the+immature+stages+of+resistant+and+susceptible+strains+of+some+common+stored+product+beetles,+and+implications+for+their+control.">The toxicity of phosphine to the immature stages of resistant and susceptible strains of some common stored product beetles, and implications for their control.</a> J. Stored Prod. Res. 24, 51-59 (1988).</li><li>Robertson, J. L., Preisler, H. K., Russel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PoloPlus: Probit and Logit Analysis User's Guide. , (2003).</li><li>Montes, C., Cuadrillero, C., Vilella, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+a+laboratory+colony+of+Cimex+lectularius+(Hemiptera:+Cimicidae)+using+an+artificial+feeding+technique.">Maintenance of a laboratory colony of Cimex lectularius (Hemiptera: Cimicidae) using an artificial feeding technique.</a> J. Med. Entomol. 39, 675-679 (2002).</li><li>Brogdon, W. G., McAllister, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplification+of+adult+mosquito+bioassays+through+use+of+time-mortality+determinations+in+glass+bottles.">Simplification of adult mosquito bioassays through use of time-mortality determinations in glass bottles.</a> J. Am. Mosq. Control. Assoc. 14, 159-164 (1998).</li><li>Katarzyna, K., Saddler, A., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+age+and+larval+nutrition+on+phenotypic+expression+of+insecticide-resistance+in+Anopheles+mosquitoes.">Effects of age and larval nutrition on phenotypic expression of insecticide-resistance in Anopheles mosquitoes.</a> PLoS One. 8 (3), (2013).</li><li>Outram, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+resistance+of+insect+eggs+to+sulphuryl+fluoride-II:+the+distribution+of+sulphuryl-35+S+fluoride+in+insect+eggs+after+fumigation.">Factors affecting the resistance of insect eggs to sulphuryl fluoride-II: the distribution of sulphuryl-35 S fluoride in insect eggs after fumigation.</a> J. Stored. Prod. Res. 3, 353-358 (1967).</li></ol>

The authors have nothing to disclose.

A critical step in this assay is to ensure that no eggs that are removed from a surface are damaged prior to the assay. Many insects cement their eggs to a substrate, therefore, a preliminary test may be needed to ensure that removal does not cause mortality. This test can be conducted with several replications of a treated group (bed bugs removed from filter paper) compared to a control group (bed bugs not removed). Similar low rates of mortality (or no mortality) between the treated and control group will ensure that removal did not cause egg mortality. Thus, any mortality recorded for the insecticide assay should be due to the insecticide treatment. The same preliminary experiment can be conducted using first instars to ensure that methods for collecting do not kill the experimental insects. All experiments conducted later should include control replications that do not receive insecticide but are treated with the solvent (for these experiments, tap water was the solvent). 
Preliminary experiments should be conducted with the recommended label rate for formulated products and then serially diluted to determine the correct concentrations to produce a concentration response curve of mortality ranging from 20-80%. Previous insecticide studies on the same insects or compounds may also be insightful when trying to determine the appropriate concentration. Concentrations will need to be modified dependent upon the insect, life stage, and compound used. New solutions should be made each time an experiment is conducted. 
The exposure of first instars to wet residues was necessary to get high mortality. Although this life stage is considered more susceptible to insecticides than older nymphs and adults, high mortality was still difficult to obtain with formulated products. Submerging the first instars into insecticides similar to the eggs was not plausible because of the resultant high mortality, thus this was a limitation to the study. The alternative first instar bioassay was manipulated to maintain first instars on surfaces to force constant contact with the treatment.
Dry residue assays and bottle assays commonly used for insecticide efficacy and resistance studies do not provide methodology for testing against multiple insect life stages17. These assays are often used to determine resistance in insects, and not to test insecticide efficacy. There are limited studies on egg dipping toxicological studies and none that have reported a standard methodology previously that can be repeated for multiple insect species.
This study provides a protocol for testing insecticide efficacy against bed bug eggs and first instars. Efficacy should be evaluated in different life stages, if not all, for a more holistic view of the efficacy of a treatment program. This consideration is important because an insect's susceptibility to insecticides changes with age and development 18. While age is an important factor in insecticide susceptibility, a factor in egg insecticide susceptibility is the eggshell. The eggshell provides a barrier to insecticides and makes insect eggs particularly difficult to control19. 
These toxicological assays provide researchers a way to evaluate insecticide toxicity against various life stages of insects. Insecticidal solutions are prepared to evaluate mortality and to determine LC50 concentrations. While this protocol was used for formulated products, the steps can be manipulated for technical grade compounds and for a variety of insects and life stages to evaluate toxicity.

Bed bugs are a significant urban pest that can cause blood loss, skin irritations, sleeplessness, depression, and anxiety in their human hosts 1,2,3,4,5,6. The costs of eliminating and controlling bed bugs are high and often times require multiple visits to a home by a pest control company7. Multiple visits are usually required because of the cryptic behavior of bed bugs and the difficulties associated with killing them. In particular, bed bug eggs are difficult to control because of their small size and the protection of the embryo by the eggshell.
Currently, a common practice for many pest control companies throughout the United States is to treat a home for bed bugs using a chemical insecticide. It is well known that the eggs are difficult to control, so many companies have implemented a two-week time frame for re-treatments8. This allows bed bug eggs enough time to hatch, so that the first instar will emerge and purportedly be easier to kill with insecticides. However, there is a dearth in studies evaluating the efficacy of liquid insecticides against bed bug eggs and first instars.
Insect eggs have been documented to be the most difficult life stage to control in urban insect pests, other than bed bugs, in addition to many agricultural pests. Most of these difficulties have been attributed to the eggshell, while a few studies have reported insecticide resistance. Resistance in the egg stage has been documented in Triatoma infestans9, Pediculus humanus capitis10, Plutella xylostella11, Rhyzopertha dominica12, Cimex lectularius13, and multiple stored product beetles (i.e. Oryzaephilus surinamensis, Tribolium castaneum, Cryptolestes ferrugineus and Rhyzopertha dominica).14The development of resistance in the egg stage and immature stages, as well as complications associated with insecticide penetration through the eggshell, necessitates the need for insecticide efficacy bioassays against these life stages.
This protocol provides a step-by-step procedure for determining the efficacy of insecticides in the egg and first instar stage of common bed bugs, Cimex lectularius. Both of these protocols are concentration-response assays to allow quantification of LC50 values. Concentration-response assays are commonly used for toxicological studies, however this protocol has been adapted for easily treating groups of bed bug eggs and first instars. These assays can be adapted for various insect species&#39; eggs and immature life stages.

<table border="0" cellpadding="0" cellspacing="0" width="1067">
	<tbody>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific Inc.</td>
			<td>08-757-105</td>
			<td>Plastic</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1001-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Featherweight forceps</td>
			<td>Fisher Scientific Inc.</td>
			<td align="right">4750</td>
			<td></td>
		</tr>
		<tr>
			<td>Temprid SC (imidacloprid/bifenthrin)</td>
			<td>Bayer CropScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transport GHP (acetamiprid/bifenthrin)</td>
			<td>FMC Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td></td>
			<td>06-443-18</td>
			<td>50 mL; flat-top threaded</td>
		</tr>
		<tr>
			<td>Fine mesh</td>
			<td></td>
			<td>7250C</td>
			<td></td>
		</tr>
		<tr>
			<td>KimWipe</td>
			<td></td>
			<td>06-666A</td>
			<td>11 cm X 21 cm</td>
		</tr>
		<tr>
			<td>Small paint brush</td>
			<td></td>
			<td></td>
			<td>Any small paint brush</td>
		</tr>
		<tr>
			<td>Hardboard panels</td>
			<td></td>
			<td></td>
			<td>Composite wood or tile would be sufficient from a home improvement store</td>
		</tr>
		<tr>
			<td>Metal weights</td>
			<td></td>
			<td></td>
			<td>Any type of metal screw that will hold the weight of the Petri dish down onto a surface</td>
		</tr>
	</tbody>
</table>

a method for evaluating insecticide efficacy against bed bug, cimex lectularius, eggs and first instars

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

The eggs were dipped into 5 different concentrations of imidacloprid/ &#946;-cyfluthrin (&#181;L/mL). The first instars were placed onto treated surfaces of five different concentrations of imidacloprid/&#946;-cyfluthrin (Figure 2).
We used three different populations of C. lectularius: Harlan, Richmond, and Epic Center (Table 1). The Harlan strain is susceptible to pyrethroid insecticides and was acquired in 2005 from Dr. Harold Harlan. The Richmond and Epic Center strains are both resistant to deltamethrin. The Richmond strain was collected from an elderly group home in Richmond, VA in 2008. The Epic Center strain was collected in 2008 in Cincinnati, OH.
Higher LC50 values indicated that a higher concentration was required to kill 50% of the test population eggs from Richmond and Epic Center strains. Similar to the eggs, the first instars from the Richmond and Epic Center populations required higher concentrations of insecticide to kill 50% of the test population. The LC50 values followed by different letters are significantly different.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55092/55092fig1.jpg" /> 
Figure 1. Photograph of the modified centrifuge tube. A hole has been cut in the cap, so that a square piece of mesh can be screwed into the cap. The mesh pieces can be discarded and replaced when using different insecticides. The bed bug eggs are placed directly onto the mesh using a paint brush and then submerged into the insecticide solutions for the dipping assay. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55092/55092fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55092/55092fig2.jpg" /> 
Figure 2. Representation of a dose-response graph. The percent response (mortality) of Harlan strain bed bug eggs to imidacloprid/&#946;-cyfluthrin is plotted on the y-axis and the log concentration that the bed bugs were exposed to is represented on the x-axis. Egg mortality increased as the dose increased and mortality reached 100% at the highest concentration tested.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Life Stage/Strain</td>
			<td colspan="2">n</td>
			<td>LC50 (95% CI)</td>
			<td>Slope &#177; SE</td>
			<td>X2 (df)</td>
		</tr>
		<tr>
			<td>Eggs</td>
			<td colspan="2"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Harlan</td>
			<td colspan="2">250</td>
			<td>0.41a (0.276-0.548)</td>
			<td>1.86 &#177; 0.24</td>
			<td>33.42 (23)</td>
		</tr>
		<tr>
			<td>Richmond</td>
			<td colspan="2">320</td>
			<td>1.23b (0.59-2.10)</td>
			<td>1.13 &#177; 0.14</td>
			<td>82.57 (30)</td>
		</tr>
		<tr>
			<td>Epic Center</td>
			<td colspan="2">400</td>
			<td>2.10b (1.049-4.587)</td>
			<td>0.95 &#177; 0.10</td>
			<td>149.91 (38)</td>
		</tr>
		<tr>
			<td>First Instars</td>
			<td colspan="2"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Harlan</td>
			<td colspan="2">150</td>
			<td>0.04a (0.030-0.063)</td>
			<td>2.16 &#177; 0.34</td>
			<td>38.17 (28)</td>
		</tr>
		<tr>
			<td>Richmond</td>
			<td colspan="2">195</td>
			<td>4.81b (1.94-10.26)</td>
			<td>0.66 &#177; 0.12</td>
			<td>45.87 (37)</td>
		</tr>
		<tr>
			<td>Epic Center</td>
			<td colspan="2">190</td>
			<td>19.72b (8.18-184.48)</td>
			<td>0.75 &#177; 0.17</td>
			<td>45.39 (36)</td>
		</tr>
	</tbody>
</table>
Table 1. Comparison of bed bug egg and first instar LC50 values modified from Campbell and Miller 2015 13.

Watch this Scientific Journal Video about A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars at JoVE.com

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

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

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

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

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Nanyang Technological University

Research

JoVE Journal

Environment

11.2K Views.  University of Florida. Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages. 

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

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Image-based Lagrangian Particle Tracking in Bed-load Experiments

Image analysis has been increasingly used for the measurement of river flows due to its capabilities to furnish detailed quantitative depictions at a relatively low cost. This manuscript describes an application of particle tracking velocimetry (PTV) to a bed-load experiment with lightweight sediment. The key characteristics of the investigated sediment transport conditions were the presence of a covered flow and of a fixed rough bed above which particles were released in limited number at the flume inlet. Under the applied flow conditions, the motion of the individual bed-load particles was intermittent, with alternating movement and stillness terms. The flow pattern was preliminarily characterized by acoustic measurements of vertical profiles of the stream-wise velocity. During process visualization, a large field of view was obtained using two action-cameras placed at different locations along the flume. The experimental protocol is described in terms of channel calibration, experiment realization, image pre-processing, automatic particle tracking, and post-processing of particle track data from the two cameras. The presented proof-of-concept results include probability distributions of the particle hop length and duration. The achievements of this work are compared to those of existing literature to demonstrate the validity of the protocol.

The manuscript presents a protocol for the conduction of bed-load sediment transport experiments where the moving particles are tracked by image analysis. The experimental facility, the procedures for run realization and data processing, and finally some proof-of-concept results are presented here.

Image analysis has been increasingly used for the measurement of river flows due to its capabilities to furnish detailed quantitative depictions at a ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform across ecological zones. To best protect public and veterinary health from these insects, the environmental limitations of pesticides need to be investigated to inform effective use of the most appropriate pesticide formulations and techniques. We have developed a research program to evaluate combinations of pesticides, pesticide application equipment, and application techniques in hot-arid desert, hot-humid tropical, warm and cool temperate, and urban locations to derive pesticide use guidelines specific to target insect and environment. To these ends we designed a system of protocols to support efficient, cost-effective, portable, and standardized evaluation of a diverse range of pesticides and equipment across multiple environments. At the core of these protocols is the use of an array of small cages with colony-reared sentinel mosquitoes (adults and immatures) and sand flies (adults), strategically arranged in natural habitats and exposed to pesticide spray. Spatial and temporal patterns of pesticide efficacy are derived from percent mortality in sentinel cages, then mapped and visualized in a geographic information system. Maps of sentinel mortality data may be statistically compared to evaluate relative efficacy of a pesticide across multiple environments, or to study multiple pesticides in a single environment. Protocols may be modified to accommodate a variety of scenarios, including, for example, the vertical orientation of sentinels in canopy habitats or simultaneous testing of ground and aerial application methods.

The efficacy of public health pesticides targeting nuisance and disease-vector insects is not uniform across different ecological zones. Here we present a system of techniques using captive vector insects as sentinels for pesticide efficacy to derive electronic maps supporting the standard evaluation of pesticides across multiple environments.

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform ...