The authors express their thanks to the National Federation of Coffee Growers of Colombia, the assistants of the Department of Entomology (Diana Marcela Giraldo, Gloria Patricia Naranjo), Experiment Station Naranjal, and Jhon F&#233;lix Trejos.

NOTE: This protocol addresses different methods to identify different effects on the CBB under laboratory conditions.
1. Fruit collection
<ol>
	<li>Pick GFs with a developmental age of ~120-150 days after flowering from trees in a coffee plantation early in the morning.</li>
</ol>
2. Fruit disinfection20
<ol>
	<li>Bring around 300 GFs to the laboratory. Select uniformly sized and healthy GFs and withdraw the peduncles.</li>
	<li>Dip the GFs into a soap solution (2 mL of liquid dishwashing soap in 998 mL of tap water), followed by rubbing to wash the GFs. Then, rinse the fruits with water, changing the water three times.</li>
	<li>Immerse GFs in 0.5% sodium hypochlorite solution (100 mL in 900 mL of tap water) and stir in a shaker at 110 rpm for 15 min. Then, rinse the GFs with water by stirring in a shaker and changing the water three times, every 10 min.</li>
	<li>Dry the GFs with sterile paper towels.</li>
	<li>Place the GFs in trays (33 cm x 25 cm x 2 cm) and irradiate them for 15 min, placing the GFs at a distance of 55 cm from the UV source inside a UV-enabled horizontal laminar flow station.</li>
	<li>During the 15 min period, every 5 min, move the GFs to ensure irradiation of the whole fruit.</li>
</ol>
3. Insect disinfection21
<ol>
	<li>Use newly emerged (same-day) CBB insects to set up the bioassays.</li>
	<li>Immerse the CBBs in 0.5% sodium hypochlorite solution, agitating them slowly with a brush for 10 min.</li>
	<li>Filter the CBBs through a muslin cloth and wash them three times with sterile distilled water.</li>
	<li>Remove excess water with sterile paper towels.</li>
</ol>
4. Evaluation of a product with a protective effect on the fruits (preinfestation) (Figure 1)
<ol>
	<li>Use a group of GFs per experimental unit. Generally, a group of 30 GFs are used per experimental unit.</li>
	<li>Place the GFs in plastic boxes (experimental unit).</li>
	<li>Apply the test product at the different concentrations for evaluation. Carry out the application with a portable sprayer unit. Here an alkaloid emulsion at 5% and 6% were tested.</li>
	<li>As a control, spray a group of GFs with water.</li>
	<li>Utilize at least three repetitions (experimental unit) per treatment, spraying one after the other.</li>
	<li>In a sterile hood, release two CBB adults per GFs (a total of 60 CBBs are introduced into the plastic boxes). After 30 min, cover the boxes.</li>
	<li>Leave the plastic boxes with the infested GFs in a room or incubator under controlled conditions (dark, 25 &#177; 2 &#176;C, and relative humidity 71% &#177; 5%).</li>
	<li>After 1, 7, 15, and 21 days, count the number of borer fruits and living and dead insects outside the fruits in each box.</li>
	<li>At 20 days postinfestation, dissect each GF under a stereomicroscope, magnification 10x.</li>
	<li>Count the number of healthy seeds or seeds damaged by the insects in each fruit.</li>
	<li>Count the different CBB biological stages22 observed and count the number of dead insects in each seed to determine insect mortality per experimental unit.</li>
</ol>
5. Evaluation of the effect of a product after CBB infestation (postinfestation) (Figure 3)
<ol>
	<li>Use groups of 200 fruits per treatment.</li>
	<li>In asterile hood,release CBB adults (2:1 ratio of CBB adults to GFs) to the previously disinfected GFs, allowing the infestation to proceed for 3 h at 21 &#176;C.</li>
	<li>Examine the GFs. After 3 h, most should be infested, with the abdomen of the CBBs still exposed (position A20), as shown in Figure 2.</li>
	<li>Select 46 infested GFs (position A) and place them in 96-well plastic racks (experimental unit). The fruits should remain in this position so that the treatment can be directly sprayed on the CBB perforating the fruit.</li>
	<li>Spray at least three time (three racks) per treatment, one after the other, covering the racks after 30 min.</li>
	<li>Leave the racks with the infested GFs in a room or incubator under controlled conditions (dark, 25 &#177; 2 &#176;C, and relative humidity 71% &#177; 5%).</li>
	<li>After 20 days, dissect the GFs under a stereomicroscope at 10x magnification.</li>
	<li>Count the number of healthy seeds or seeds damaged by the insects in each fruit.</li>
	<li>Count the different CBB biological stages22 and the number of dead insects in each seed to determine insect mortality per experimental unit.</li>
</ol>
6. Evaluation of a product with a deterrent effect on the CBB
<ol>
	<li>Follow the steps 4.1-4.6 outlined for evaluating a product with a protective effect on the fruits.</li>
	<li>After releasing the CBB adults into the plastic boxes, count the number of CBBs that fly away from the boxes and the number that infest the GFs. Then, follow steps 4.7-4.11.</li>
	<li>Follow the steps 5.1-5.5 outlined for evaluating the product after the CBB infestation.</li>
	<li>After spraying each treatment on the insects in position A, count the number of CBBs that moved out of the GF and/or flew away from the GF. Then, follow steps 5.6-5.9.</li>
</ol>
7. Statistical analysis
NOTE: The response variables are mortality percentages over time and the percentage of healthy uninfested coffee seeds.
<ol>
	<li>Estimate the average and standard deviation of each response variable for each treatment.</li>
	<li>Perform analysis of variance for each response variable with a model for a completely randomized design. 
	NOTE: Dunnett&#39;s 5% comparison test is performed to compare the treatments against the absolute control (water control).</li>
	<li>When the treatments are significantly different from the absolute control, use a 5% least significant difference (LSD) test to compare the treatments.</li>
	<li>Evaluate the power of the test; if greater than 85%, the assumptions of normality and homogeneity of the variances are met.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63694/63694fig01.jpg" /> 
Figure 1: Procedure for the evaluation of the preinfestation effects of insecticides on the CBB. Steps for evaluating the preinfestation effects of insecticides on Hypothenemus hampei (CBB) using green fruits (GFs).&#160;(A) Fruit selection. (B) Spraying of the insecticides on the coffee fruits. (C) CBB infestation of coffee fruits at a ratio of 2:1 CBB per GF. (D) Infested fruits. (E) Incubation of the fruits under controlled conditions. (F) Fruit dissection. (G) Counting the CBB population inside the seeds. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63694/63694fig01large.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/63694/63694fig02.jpg" /> 
Figure 2: Process coffee fruits&#39; CBB infestation. The infested fruits contain CBB adults with their abdomens partially exposed (position A). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63694/63694fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63694/63694fig03.jpg" /> 
Figure 3: Procedure for the evaluation of the posinfestation effects of insecticides on the CBB. Steps for evaluating the postinfestation effects of insecticides on the CBB using GFs. (A) Fruit selection. (B) Infestation of the fruits with CBB at a ratio of 2:1 CBB per GF. (C) Selection of infested fruits. (D) Spraying of the insecticide on the fruits. (E) Incubation of the fruits. (F) Fruit dissection. (G) Counting the CBB population. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63694/63694fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Mendesil, E., 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=Semiochemicals+used+in+host+location+by+the+coffee+berry+borer,+Hypothenemus+hampei.">Semiochemicals used in host location by the coffee berry borer, Hypothenemus hampei.</a> Journal of Chemical Ecology. 35 (8), 944-950 (2009).</li><li>Jaramillo, J., 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=Coffee+berry+borer+joins+bark+beetles+in+coffee+klatch.">Coffee berry borer joins bark beetles in coffee klatch.</a> PLoS ONE. 8 (9), 74277 (2013).</li><li>Giraldo-Jaramillo, M., Garcia, A. G., Parra, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology,+thermal+requirements,+and+estimation+of+the+number+of+generations+of+Hypothenemus+hampei+(Ferrari,+1867)+(Coleoptera:+Curculionidae)+in+the+state+of+S&#227;o+Paulo,+Brazil.">Biology, thermal requirements, and estimation of the number of generations of Hypothenemus hampei (Ferrari, 1867) (Coleoptera: Curculionidae) in the state of S&#227;o Paulo, Brazil.</a> Journal of Economic Entomology. 111 (5), 2192-2200 (2018).</li><li>Benavides, P., G&#243;ngora, C., Bustillo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPM+Program+to+Control+Coffee+Berry+Borer+Hypothenemus+hampei,+with+Emphasis+on+Highly+Pathogenic+Mixed+Strains+of+Beauveria+bassiana,+to+Overcome+Insecticide+Resistance+in+Colombia.">IPM Program to Control Coffee Berry Borer Hypothenemus hampei, with Emphasis on Highly Pathogenic Mixed Strains of Beauveria bassiana, to Overcome Insecticide Resistance in Colombia.</a> IntechOpen. , (2012).</li><li>Mart&#237;nez, C. P., Echeverri, C., Florez, J. C., Gaitan, A. L., G&#243;ngora, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+production+of+two+chitinolytic+proteins+with+an+inhibiting+effect+on+the+insect+coffee+berry+borer,+Hypothenemus+hampei+(Ferrari)+(Coleoptera:+Curculionidae)+and+the+fungus+Hemileia+vastatrix+the+most+limiting+pests+of+coffee+crops.">In vitro production of two chitinolytic proteins with an inhibiting effect on the insect coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) and the fungus Hemileia vastatrix the most limiting pests of coffee crops.</a> AMB Express. 2, 1-11 (2012).</li><li>Padilla, B. E., Acu&#241;a, Z., Vel&#225;squez, C. S., Rubio, G. J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+[alpha]-amylases+from+the+coffee+berry+borer+Hypothenemus+hampei+in+different+plant+species.">Inhibitors of [alpha]-amylases from the coffee berry borer Hypothenemus hampei in different plant species.</a> Revista Colombiana de Entomolog&#237;a. 32 (2), 125-130 (2006).</li><li>Alvarez, J. H., Cortina, H. A., Villegas, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+evaluate+antibiosis+to+Hypothenemus+hampei+Ferrari+in+coffee+under+controlled+conditions.">Methods to evaluate antibiosis to Hypothenemus hampei Ferrari in coffee under controlled conditions.</a> Cenicaf&#233;. 52 (3), 205-214 (2001).</li><li>. New Product in the Integrated Management of the Coffee Berry Borer in Colombia. National Coffee Research Center (Cenicaf&#233;) Available from: <a target="_blank" href="https://biblioteca.cenicafe.org/handle/10778/477">https://biblioteca.cenicafe.org/handle/10778/477</a> (2014)</li><li>Jaramillo, J., Montoya, E., Benavides, P., G&#243;ngora, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beauveria+bassiana+and+Metarhizium+anisopliae+for+the+control+of+coffee+brocade+in+fruits+on+the+ground.">Beauveria bassiana and Metarhizium anisopliae for the control of coffee brocade in fruits on the ground.</a> Revista Colombiana de Entomolog&#237;a. 41, 95-104 (2015).</li><li>Bastidas, A., Vel&#225;squez, E., Benavides, P., Bustillo, A., Orozco, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+preformulated+Beauveria+bassiana+(B&#225;lsam)+Vuillemin,+for+the+control+of+the+coffee+berry+borer.">Evaluation of preformulated Beauveria bassiana (B&#225;lsam) Vuillemin, for the control of the coffee berry borer.</a> Agronomia. 17, 44-61 (2009).</li><li>Villalba-Gault, D., Bustillo, A., Chaves Cordoba, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+insecticides+for+the+control+of+the+coffee+berry+borer+in+Colombia.">Evaluation of insecticides for the control of the coffee berry borer in Colombia.</a> Cenicafe. 46, 152-163 (1995).</li><li>Bustillo, A. E., Orozco, J., Benavides, P., Portilla, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+and+use+of+parasitoids+for+the+control+of+the+coffee+berry+borer+in+Colombia.">Mass production and use of parasitoids for the control of the coffee berry borer in Colombia.</a> Cenicafe. 47 (4), 215-230 (1996).</li><li>Celestino, F. N., Pratissoli, D., Machado, L. C., Santos Junior, H. J. G. D., Mardgan, L., Ribeiro, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+breeding+techniques+of+the+coffee+berry+borer+[Hypothenemus+hampei+(Ferrari).">Adaptation of breeding techniques of the coffee berry borer [Hypothenemus hampei (Ferrari).</a> Coffee Science. 11 (2), 161-168 (2016).</li><li>Dom&#237;nguez, L., Parzanese, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet+light+in+food+preservation.">Ultraviolet light in food preservation.</a> Argentine Foods. 52 (2), 70-76 (2012).</li><li>Jaramillo, J., Chabi-Olaye, A., Poehling, H. M., Kamonjo, C., Borgemeister, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+improved+laboratory+production+technique+for+the+coffee+berry+borer+Hypothenemus+hampei,+using+fresh+coffee+berries.">Development of an improved laboratory production technique for the coffee berry borer Hypothenemus hampei, using fresh coffee berries.</a> Entomologia Experimentalis et Applicata. 130 (3), 275-281 (2009).</li><li>P&#233;rez, J., Infante, F., Vega, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+coffee+berry+borer+(Coleoptera:+Scolytidae)+have+mutualistic+fungi.">Does the coffee berry borer (Coleoptera: Scolytidae) have mutualistic fungi.</a> Annals of the Entomological Society of America. 98 (4), 483-490 (2005).</li><li>Benavides, P., Gil, P., G&#243;ngora, C., Arcila, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+pest+management.+Cenicafe.+Manual+of+the+Colombian+coffee+grower:+Research+and+technology+for+the+sustainability+of+coffee+growing.">Integrated pest management. Cenicafe. Manual of the Colombian coffee grower: Research and technology for the sustainability of coffee growing.</a> Manizales: FNC: Cenicaf&#233;. 3, 179-214 (2013).</li><li>Bustillo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+coffee+berry+borer,+Hypothenemus+hampei+(Coleoptera:+Curculionidae:+Scolytinae),+in+Colombia.">A review of the coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae), in Colombia.</a> Revista Colombiana de Entomolog&#237;a. 32 (2), 101-116 (2006).</li><li>New Chemical Control Alternative for the Integrated Management of the Coffee Berry Borer. National Coffee Research Center (Cenicaf&#233;) Available from: <a target="_blank" href="https://biblioteca.cenicafe.org/handle/10778/557">https://biblioteca.cenicafe.org/handle/10778/557</a> (2015)</li><li>Tapias, L., Martinez, C., Benavides, P., Gongora, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+method+to+evaluate+the+effect+of+insecticides+on+the+coffee+berry+borer.">Laboratory method to evaluate the effect of insecticides on the coffee berry borer.</a> Cenicaf&#233;. 68 (2), 76-89 (2017).</li><li>Bustillo, A. E., Mar&#237;n, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+reactivate+the+virulence+of+Beauveria+bassiana+to+control+the+coffee+berry+borer.">How to reactivate the virulence of Beauveria bassiana to control the coffee berry borer.</a> Manejo Integrado de Plagas. 63, (2002).</li><li>Constantino, L. 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=morphological+and+genetic+aspects+of+Hypothenemus+obscurus+and+Hypothenemus+hampei+(Coleoptera:+Curculionidae:+Scolytinae).">morphological and genetic aspects of Hypothenemus obscurus and Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae).</a> Revista Colombiana de Entomolog&#237;a. 37 (2), 173-182 (2011).</li><li>Estrela, 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=Mechanism+of+action+of+sodium+hypochlorite.">Mechanism of action of sodium hypochlorite.</a> Brazilian Dental Journal. 13 (2), 113-117 (2002).</li><li>Diffey, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solar+ultraviolet+radiation+effects+on+biological+systems.">Solar ultraviolet radiation effects on biological systems.</a> Physics in Medicine and Biology. 36 (3), 299-328 (1991).</li><li>. Integrated Management of the Coffee Berry Borer: Hypothenemus hampei Ferrari in Colombia Available from: <a target="_blank" href="https://biblioteca.cenicafe.org/hangle/10778/848">https://biblioteca.cenicafe.org/hangle/10778/848</a> (1998)</li><li>Portilla, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+an+artificial+diet+for+the+rearing+of+Hypothenemus+hampei.">Development and evaluation of an artificial diet for the rearing of Hypothenemus hampei.</a> Cenicaf&#233;. 50, 24-38 (1999).</li><li>Portilla, R. M., Streett, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+techniques+for+automated+mass+production+of+Hypothenemus+hampei+on+the+modified+Cenibroca+artificial+diet.">New techniques for automated mass production of Hypothenemus hampei on the modified Cenibroca artificial diet.</a> Cenicaf&#233;. 57, 37-50 (2006).</li></ol>

None of the authors have any conflicts of interest to declare.

In this protocol, disinfection of the fruits as well as the insects are critical steps. When fruits from the field are used in the laboratory, they frequently show high contamination and dehydration since microorganisms and mites are present in the epidermis7,15,16. Therefore, using fruits or insects that are not disinfected will cause insect death due to contamination caused by microorganisms, such as bacteria or fungi, thus in...

The coffee berry borer (CBB), Hypothenemus hampei, was first detected in 1988 in Colombia and has since become the most important pest species of the coffee crop. CBB females leave the natal fruit already fertilized, seeking new fruits guided by the volatile chemicals that they emit1,2. A complete cycle is fulfilled within 23 days3 at a temperature of 25 &#176;C. The cycle starts with the founder female penetrating the seed and laying eggs in the fruit endosperm. The eclosed larvae eat the seed. If the fruits are dissected at this point, it would be possible to observe both the founder female and her offspring. After 14&#160;days, the larvae become pupae-generally, the pupae stage lasts 5 days. In the adult stage, the females copulate with their siblings, and the newly fertilized females fly away from the damaged fruits looking for new coffee fruits to start a new cycle4.
Both the penetration process and the result of larval feeding damage the coffee seed, decreasing the quality of the coffee beverage and significantly reducing the revenue; greater than 5% infestation in coffee plantations is generally considered the economic threshold.
CBB control is based on an integrated pest management (IPM) strategy, including cultural control and agronomic practices, natural biological agents, and the use of chemical insecticides, which requires safety conditions and timely application4.
To evaluate new insecticides for the control of the CBB, low-cost methodologies are needed that allow rapid results to be obtained. Both laboratory and field procedures are currently in use, including artificial diets containing coffee in which the insecticides are incorporated5,6,&#160;or spraying the insecticides on dry parchment coffee7,8,9. In addition, experiments carried out in the field using coffee tree branches covered with entomological sleeves have been reported10,11; however, these methods require intense labor and long evaluation periods.
A condition resembling natural field conditions, that is also fast and inexpensive, is the use of green or ripe coffee fruits. However, these fruits must be maintained under conditions suitable for developing the CBB, avoiding alterations and contaminants by microorganisms to maintain their quality and properties. To this end, different disinfectants have been used, as well as procedures involving heat and radiation7,9,12,13,14,15,16.
Additionally, the methods for insecticide evaluation against the CBB require simulations of adult females flying in search of fruits or penetrating those fruits17,18. For this, artificial fruit infestations have been carried out in the field8,11,19, although this process is labor-intensive and depends on environmental conditions.
Here, we describe a standardized methodology for the evaluation of products that can have different effects on the CBB under controlled environmental conditions that resemble field conditions.

<table><tbody><tr><td>Beaker with spout, low form 500 mL</td><td>BRAND PP</td><td>BR87826</td><td></td></tr><tr><td>Benchtop Shaker</td><td>New Brunswick Scientific Innova 4000 Incubator Shaker</td><td></td><td></td></tr><tr><td>Dishwashing liquid soap-AXION</td><td>Colgate-Palmolive</td><td>AXION</td><td></td></tr><tr><td>Hood; Horizontal Laminar Flow Station</td><td>Terra Universal</td><td></td><td>&amp;nbsp;Powder-Coated Steel, 1930 mm W x 1118 mm D x 1619 mm H, 120 V (https://www.terrauniversal.com/hood-horizontal-laminar-flow-station-9620-64a.html)</td></tr><tr><td>Insects CBB</td><td>BIOCAFE</td><td></td><td>(http://avispitas.blogspot.com/p/biocafe.html).</td></tr><tr><td>Multi Fold White paper towels</td><td>Familia</td><td>73551</td><td></td></tr><tr><td>Preval Spray unit</td><td>&amp;nbsp;Preval Merck</td><td>Z365556-1KT</td><td>https://www.sigmaaldrich.com/CO/es/product/sigma/z365556?gclid=Cj0KCQiAweaNBhDEARIsAJ&lt;br/&gt; 5hwbfZOy1TWGj6huatFtRQt&lt;br/&gt; AzOyHe5-oBiKnOUK2T1exuuk&lt;br/&gt; WwJLdvxkvsaAjoYEALw_wcB</td></tr><tr><td>Reversible Racks 96-Well</td><td>heathrowscientific</td><td>HEA2345A</td><td>https://www.heathrowscientific.com/reversible-racks-96-well-i-hea2345a</td></tr><tr><td>Scalpel blades N 11</td><td>Merck</td><td>S2771-100EA</td><td></td></tr><tr><td>Scalpel handles N3</td><td>Merck</td><td>S2896-1EA</td><td></td></tr><tr><td>Sodium Hypochloride</td><td>The clorox company</td><td>Clorox</td><td></td></tr><tr><td>Stereo Microscope</td><td>Zeiss</td><td>Stemi 508</td><td>https://www.zeiss.com/microscopy/int/products/stereo-zoom-microscopes/stemi-508.html</td></tr></tbody></table>

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.

The results showed that the CBB females recognized the fruits, and depending on the characteristics of the fruit surface and the emitted odors, the CBB females started to penetrate or bore the fruits within 3 h at 21 &#176;C.
The effect of an insecticide on the CBB when applied to the coffee fruits (preinfestation procedure)&#160;after 24 h and over time is shown in Figure 4. The two insecticides (alkaloid emulsion at 5% and 6%) caused high insect mortality on Day...

Watch this Scientific Journal Video about Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei at JoVE.com

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.

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 ...

methodology-to-test-control-agents-insecticides-against-coffee-berry

Research

JoVE Journal

Environment

1.8K Views.  National Coffee Research Center. 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.

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

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 ...

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p&#8804;0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

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.

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.

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

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Biochemistry

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

Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

As a vital vector of dengue fever, yellow fever, and other mosquito-borne diseases, Aedes albopictus (Diptera: Culicidae) can be broadly distributed worldwide and cause a severe threat to public health. To date, considering the fast-emerging insecticide resistance in the mosquito, the development of new botanical insecticides to control and reduce Ae. albopictus is urgent and crucial. Therefore, to investigate the decoction effect of the plant C. abrotanoides L. on mosquito larvae killing, we detected the mortality of larvae after treatment with different concentrations (60 mg/mL, 120 mg/mL, and 180 mg/mL) of decoction within a series of time points (12 h, 24 h, 36 h, and 48 h). We found that 24 h with 180 mg/mL C. abrotanoides L. decoction treatment killed 92.35% of mosquitoes relative to the control treatment. Meanwhile, 36 h with 120 mg/mL could also kill more than 90% of mosquitoes. Furthermore, Carassius auratus populations were exposed to 120 mg/mL C. abrotanoides L. decoction for 36 h and were not dead. The mortality evaluation indicated that this concentration is not a harmful level of ecological environmental pollution. This study provides a possible plant candidate that could be used for designing plant-derived insecticides. Additionally, these methods can be altered and applied to other mosquito species.

Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

Using the Carpesium abrotanoides L. plant for mosquito larvae control can effectively reduce the Aedes albopictus mosquito population and provide a foundation for designing plant-derived insecticides.

As a vital vector of dengue fever, yellow fever, and other mosquito-borne diseases, Aedes albopictus (Diptera: Culicidae) can be broadly distributed ...

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 ...

The Mushing, Sieving, and Floating Method for Detecting Fruit Fly Larvae

Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field

Fruit flies of the Tephritidae family are among the most destructive and invasive agricultural pests in the world. Many countries undertake expensive eradication programs to eliminate incipient populations. During eradication programs, a concerted effort is made to detect larvae, as this strongly indicates a breeding population and helps establish the spatial extent of the infestation. The detection of immature life stages triggers additional control and regulatory actions to contain and prevent any further spread of the pest. Traditionally, larval detection is accomplished by cutting individual host fruits and examining them visually. This method is labor intensive, as only a limited number of fruit can be processed, and the probability of missing a larva is high. An extraction technique that combines i) mushing host fruit in a plastic bag, ii) straining pulp through a series of sieves, iii) placing retained pulp in a brown sugar water solution, and iv) collecting larvae that float to the surface was tested. The method was evaluated in Florida with field-collected guava naturally infested by Anastrepha suspensa. To mimic low populations more representative of a fruit fly eradication program, mangos and papaya in Hawaii were infested with a known, low number of Bactrocera dorsalis larvae. The applicability of the method was tested in the field on guava naturally infested by B. dorsalis to evaluate the method under conditions experienced by workers during an emergency fruit fly program. In both field and laboratory trials, mushing and sieving the pulp was more efficient (required less time) and more sensitive (more larvae found) than cutting fruit. Floating the pulp in brown sugar water solution helped detect earlier instar larvae. Mushing and sieving fruit pulp of important tephritid hosts may increase the probability of detecting larvae during emergency programs.

Author Spotlight: Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field  

Increasing the detection of immature tephritid fruit flies in the field can trigger timely efforts to eliminate populations of these destructive pests. Detecting late instar larvae is faster and more accurate when mushing host fruit in a bag and passing the pulp through a series of sieves than hand cutting and visual inspection.

Fruit flies of the Tephritidae family are among the most destructive and invasive agricultural pests in the world. Many countries undertake expensive ...

Topical Testing Assay: A Technique to Evaluate the Insecticidal Potential of Dopamine Receptor Antagonists on Adult Female Mosquitoes by Topical Application

Source: Brito-Sierra, C.A. et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/57768">Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes.</a> J. Vis. Exp. (2019).
In this video, we demonstrate the topical testing assay to determine the toxicity of a potential insecticide on adult female mosquitoes. Using a dopamine receptor antagonist that causes lethal inhibition of neurotransmission in mosquitoes, this method describes a way to evaluate the toxic effects of insecticides upon topical application.

Source: Brito-Sierra, C.A. et al., Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes. J. Vis. Exp. (2019).
In this video, ...

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

Summary

Explore More Videos

Chapters in this video

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

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

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

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Bioassays for Monitoring Insecticide Resistance

Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

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

Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field

Topical Testing Assay: A Technique to Evaluate the Insecticidal Potential of Dopamine Receptor Antagonists on Adult Female Mosquitoes by Topical Application