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><li>Sabou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bed+bugs+reproductive+life+cycle+in+the+clothes+of+a+patient+suffering+from+Alzheimer%27s+disease+results+in+iron+deficiency+anemia%5BTitle%5D)+AND+%22Parasite%22%5BJournal%5D)">Bed bugs reproductive life cycle in the clothes of a patient suffering from Alzheimer's disease results in iron deficiency anemia.</a> Parasite. 20, (2013).</li>
<li>Reinhardt, K., Kempke, D., Naylor, R. A., Siva-Jothy, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitivity+to+bites+by+the+bedbug%2C+Cimex+lectularius%5BTitle%5D)+AND+%22Med.+Vet.+Entomol%22%5BJournal%5D)">Sensitivity to bites by the bedbug, Cimex lectularius.</a> Med. Vet. Entomol. 23, 163-166 (2009).</li>
<li>Leverkus, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bullous+allergic+hypersensitivity+to+bed+bug+bites+mediated+by+IgE+against+salivary+nitrophorin%5BTitle%5D)+AND+%22J.+invest.+entomol%22%5BJournal%5D)">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/pubmed?term=((Widespread+bullous+eruption+due+to+multiple+bed+bug+bites%5BTitle%5D)+AND+%22Clin.+Exp.+Dermatol%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2734207/">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/pubmed?term=((Psychological+effects+of+bed+bug+attacks+%28Cimex+lectularius+L%29%5BTitle%5D)+AND+%22Am.+J.+Med%22%5BJournal%5D)">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="https://www.researchgate.net/publication/263133743_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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPinto%2C+LJ+%22Bed+Bug+Handbook%3A+The+Complete+Guide+to+Bed+Bugs+and+Their+Control%22">Bed Bug Handbook: The Complete Guide to Bed Bugs and Their Control</a>. , Pinto & Associates, Inc.. Mechanicsville, MD, USA. (2007).</li>
<li>Toloza, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+patterns+of+insecticide+resistance+in+eggs+and+first+instars+of+Triatoma+infestans+%28Hemiptera%3A+Reduviidae%29+from+Argentina+and+Bolivia%5BTitle%5D)+AND+%22J.+Med.+Entomol%22%5BJournal%5D)">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/pubmed?term=((Evidence+of+pyrethroid+resistance+in+eggs+of+Pediculus+humanus+capitis+%28Phthiraptera%3A+Pediculidae%29+from+Argentina%5BTitle%5D)+AND+%22J.+Med.+Entomol%22%5BJournal%5D)">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/pubmed?term=((Deltamethrin+as+a+potential+ovicidal+pyrethroid+against+Plutella+xylostella+L%5BTitle%5D)+AND+%22Toxicol.+let%22%5BJournal%5D)">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. The occurrence of resistance to phosphine in adult and egg stages of strains of Rhyzopertha dominica (F.)(Coleoptera: Bostrichidae). 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/pubmed?term=((Insecticide+Resistance+in+Eggs+and+First+Instars+of+the+Bed+Bug%2C+Cimex+lectularius+%28Hemiptera%3A+Cimicidae%29%5BTitle%5D)+AND+%22Insects%22%5BJournal%5D)">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="https://www-sciencedirect-com.remotexs.ntu.edu.sg/science/article/pii/0022474X88900082">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRobertson%2C+JL+%22PoloPlus%3A+Probit+and+Logit+Analysis+User%27s+Guide%22">PoloPlus: Probit and Logit Analysis User's Guide</a>. , LeOra Software. Petaluna, CA, USA. (2003).</li>
<li>Montes, C., Cuadrillero, C., Vilella, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maintenance+of+a+laboratory+colony+of+Cimex+lectularius+%28Hemiptera%3A+Cimicidae%29+using+an+artificial+feeding+technique%5BTitle%5D)+AND+%22J.+Med.+Entomol%22%5BJournal%5D)">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/pubmed?term=((Simplification+of+adult+mosquito+bioassays+through+use+of+time-mortality+determinations+in+glass+bottles%5BTitle%5D)+AND+%22J.+Am.+Mosq.+Control.+Assoc%22%5BJournal%5D)">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/pubmed?term=((Effects+of+age+and+larval+nutrition+on+phenotypic+expression+of+insecticide-resistance+in+Anopheles+mosquitoes%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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="https://www.researchgate.net/publication/248283593_Factors_affecting_the_resistance_of_insect_eggs_to_sulphuryl_fluoride-I_The_uptake_of_sulphuryl-35S_fluoride_by_insect_eggs">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><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>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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11.3K 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 Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene Md&#945;E7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (Md&#945;E7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of Md&#945;E7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of Md&#945;E7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

Biochemistry

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these insecticides against adult insects or their impact on reproductive output. However, currently available methods assess adult mortality only, limiting the selection of novel insecticides with a different mode of action. In this work, different experimental methods were examined to identify the diverse effects on the CBB under laboratory conditions. For this, green coffee fruits (GFs) were collected and disinfected by immersion in sodium hypochlorite solution followed by UV light irradiation. In parallel, CBB adults from a colony were disinfected by immersion in sodium hypochlorite solution. To assess fruit protection (preinfestation), the fruits were placed in plastic boxes, and the insecticides were applied. Then, the CBB adults were released at a rate of two CBBs per GF. The GFs were left under controlled conditions to evaluate CBB infestation and survival after 1, 7, 15, and 21 days. To evaluate insecticide efficacy after CBB infestation (postinfestation), CBB adults were released to the GFs in a 2:1 ratio for 3 h at 21 &#176;C. Infested fruits showing CBB adults with their abdomens partially exposed were selected and placed in 96-well racks, and the CBBs boring into the fruits were treated directly. After 20 days, the fruits were dissected, and the CBB biological stages inside each fruit were recorded. The GFs served as substrates that mimic natural conditions to evaluate toxic, chemical, and biological insecticides against the CBB.

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

A method using green coffee fruits (GFs) was developed to test the toxicity of insecticides against the coffee berry borer (CBB). Insecticides or toxic substances were applied to disinfected GFs before or after CBB infestation. Insect mortality, repellency, and reproductive capacity, in addition to other parameters, were evaluated.

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these ...

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ingestion of provisions contaminated with pesticides may increase the mortality rate of the larvae, decrease the collection rate and the population of adult solitary bees in the next year from a demographic perspective. But there are limited studies on the effects of pesticides on the larvae of solitary bees. Therefore, understanding how pesticides influence the larvae of solitary bees should be considered an integral part of pesticide ecological risk assessment. This study presents a method to expose the larvae of solitary bee, Osmia excavata, to lethal or sublethal doses of pesticide, tracking larval weight gain, developmental duration, eclosion ability, and food consumption efficiency conversion of ingested food. To demonstrate the effectiveness of this method, the larvae of O. excavata were fed with provisions containing acute lethal and sublethal doses of chlorpyrifos. Then, the above indexes of the treated larvae were investigated. This technique helps to predict and mitigate the risk of pesticides to pollinators.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

Bioassays for Monitoring Insecticide Resistance

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

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

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

Biology

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

Neuroscience

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

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

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

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

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

Assessment of Phytochemical Effects on Helicoverpa armigera Through Feeding Assay

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

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

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

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

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

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

Source: Lee, S. Y. and Kang, K. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2017).
C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.&#160; This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Source: Lee, S. Y. and Kang, K. Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. 
...

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

Summary

Explore More Videos

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Bioassays for Monitoring Insecticide Resistance

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

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

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

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans