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

Summary

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.

Abstract

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 °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.

Introduction

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 emit^1,2. A complete cycle is fulfilled within 23 days³ at a temperature of 25 °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 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 incorporated^5,6, or spraying the insecticides on dry parchment coffee^7,8,9. In addition, experiments carried out in the field using coffee tree branches covered with entomological sleeves have been reported^10,11; 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 radiation^{7,9,12,13,14,15,16}.

Additionally, the methods for insecticide evaluation against the CBB require simulations of adult females flying in search of fruits or penetrating those fruits^17,18. For this, artificial fruit infestations have been carried out in the field^8,11,19, 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.

Protocol

NOTE: This protocol addresses different methods to identify different effects on the CBB under laboratory conditions.

1. Fruit collection

1. Pick GFs with a developmental age of ~120-150 days after flowering from trees in a coffee plantation early in the morning.

2. Fruit disinfection²⁰

1. Bring around 300 GFs to the laboratory. Select uniformly sized and healthy GFs and withdraw the peduncles.
2. 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.
3. 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.
4. Dry the GFs with sterile paper towels.
5. 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.
6. During the 15 min period, every 5 min, move the GFs to ensure irradiation of the whole fruit.

3. Insect disinfection²¹

1. Use newly emerged (same-day) CBB insects to set up the bioassays.
2. Immerse the CBBs in 0.5% sodium hypochlorite solution, agitating them slowly with a brush for 10 min.
3. Filter the CBBs through a muslin cloth and wash them three times with sterile distilled water.
4. Remove excess water with sterile paper towels.

4. Evaluation of a product with a protective effect on the fruits (preinfestation) (Figure 1)

1. Use a group of GFs per experimental unit. Generally, a group of 30 GFs are used per experimental unit.
2. Place the GFs in plastic boxes (experimental unit).
3. 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.
4. As a control, spray a group of GFs with water.
5. Utilize at least three repetitions (experimental unit) per treatment, spraying one after the other.
6. 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.
7. Leave the plastic boxes with the infested GFs in a room or incubator under controlled conditions (dark, 25 ± 2 °C, and relative humidity 71% ± 5%).
8. After 1, 7, 15, and 21 days, count the number of borer fruits and living and dead insects outside the fruits in each box.
9. At 20 days postinfestation, dissect each GF under a stereomicroscope, magnification 10x.
10. Count the number of healthy seeds or seeds damaged by the insects in each fruit.
11. Count the different CBB biological stages²² observed and count the number of dead insects in each seed to determine insect mortality per experimental unit.

5. Evaluation of the effect of a product after CBB infestation (postinfestation) (Figure 3)

1. Use groups of 200 fruits per treatment.
2. 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 °C.
3. 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.
4. 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.
5. Spray at least three time (three racks) per treatment, one after the other, covering the racks after 30 min.
6. Leave the racks with the infested GFs in a room or incubator under controlled conditions (dark, 25 ± 2 °C, and relative humidity 71% ± 5%).
7. After 20 days, dissect the GFs under a stereomicroscope at 10x magnification.
8. Count the number of healthy seeds or seeds damaged by the insects in each fruit.
9. Count the different CBB biological stages²² and the number of dead insects in each seed to determine insect mortality per experimental unit.

6. Evaluation of a product with a deterrent effect on the CBB

1. Follow the steps 4.1-4.6 outlined for evaluating a product with a protective effect on the fruits.
2. 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.
3. Follow the steps 5.1-5.5 outlined for evaluating the product after the CBB infestation.
4. 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.

7. Statistical analysis

NOTE: The response variables are mortality percentages over time and the percentage of healthy uninfested coffee seeds.

1. Estimate the average and standard deviation of each response variable for each treatment.
2. Perform analysis of variance for each response variable with a model for a completely randomized design.
   NOTE: Dunnett's 5% comparison test is performed to compare the treatments against the absolute control (water control).
3. When the treatments are significantly different from the absolute control, use a 5% least significant difference (LSD) test to compare the treatments.
4. Evaluate the power of the test; if greater than 85%, the assumptions of normality and homogeneity of the variances are met.

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). (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. Please click here to view a larger version of this figure.

Figure 2: Process coffee fruits' CBB infestation. The infested fruits contain CBB adults with their abdomens partially exposed (position A). Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Representative Results

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 °C.

The effect of an insecticide on the CBB when applied to the coffee fruits (preinfestation procedure) 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 20 (Table 1) and showed significant differences compared to the water absolute control (P < 0.001), according to the LSD test. Regarding the percentages of uninfested healthy seeds (Table 1), there were also differences between the control and the insecticides groups according to Dunnett's test at 5% (P < 0.001). In the control group, 37% of the seeds were not infested, while insecticides application protected the seeds, with 94% of the seeds remaining healthy when using insecticide 2 and 89% with insecticide 1.

Figure 4: Preinfestation effects of insecticides in control vs. two insecticide groups. Preinfestation effects of the insecticides. Percent mortalityof adult H. hampei evaluated on days 1, 7, 15, and 21 after infestation. Please click here to view a larger version of this figure.

Treatment	Experimental unit	Mortality (%)			Healthy seed (%)
		Average		Sd	Average		Sd
Control (water)	5	12.4		8.3	37		6.3
Insecticide 1	5	83.9	*b	3.9	89	*b	6
Insecticide 2	5	94.2	*a	3.2	94.2	*a	3.7
* For each variable, differences with respect to the control (water) according to Dunnett's test at 5%.

Table 1: Effect of preinfestation treatment on the CBB. Percent mortality and percenthealthy seeds after 20 days. * For each variable, differences with respect to the control (water) according to Dunnett's test at 5%.

The preinfestation results after 21 days are shown in Table 1, and the results over time correspond to Figure 4. In this case, the coffee fruits were covered with a toxic substance that causes insect mortality. The insects become impregnated when they walk over the fruits, taste the fruits with their palps, or start chewing the epidermis of the fruits. Additionally, the substances applied over the fruit surface can alter or change the natural odor of the fruit, so the CBB individuals may stop the infestation process, either flying away or preferring to be separated from the fruit without touching it or infesting it. Depending on the time of action of the product, insect mortality or avoiding infestation behavior can persist for 24 h or longer.

On the other hand, if the products are applied after the insects start to bore the fruits (postinfestation), the products can penetrate the insect cuticle, causing insect mortality (Table 2 and Figure 5). The highest mortality occurred with insecticide 2 (P < 0.01). If mortality occurs rapidly, the insect will die before it enters the seed, and no eggs or insect population will be found inside the seeds.

Figure 5: Postinfestation effects of insecticides. Percent mortality of adult H. hampei evaluated on days 1, 7, 15, and 21 after infestation. Please click here to view a larger version of this figure.

Treatment	Experimental unit	Mortality (%)			Healthy seed (%)
		Average		Sd	Average		Sd
Control (water)	5	11.1		3.0	57.3		3. 9
Insecticide 1	5	46.8	*b	6.6	79.2	*b	8.6
Insecticide 2	5	77.8	*a	3.7	90.0	*a	2.9
* For each variable, differences with respect to the control (water) according to Dunnett's test at 5%.

Table 2: Effects of postinfestation treatment on the CBB. Percent mortality and percent healthy seeds after 20 days. * For each variable, differences with respect to the control (water) according to Dunnett's test at 5%.For each variable, different letters indicate differences according to LSD 5%.

The effects of the insecticides are reflected as the percentage of healthy uninfested seeds on day 20 of evaluation (Table 2). Due to high insect mortality, the insect did not penetrate the coffee seeds and damage them. Application of the products protected between 79%-90% of the coffee seeds, showing differences with respect to the control, in which 57% of the seeds were found to be healthy (P < 0.01). Significant differences were also observed between the two insecticides (P < 0.01).

In some cases, the insects died very quickly, even before causing damage to the seed. However, if the insect's death took longer, the insect could reach the seed and deposit some eggs, and later, the adult will die. In this case, a reduced insect population was found inside the coffee seeds compared with the insect population found in the control group sprayed with water (Table 3).

Treatments	Total Average Insect Population/ seed	* Duncan grouping (alpha= 00.05)
Control	5	a
Entomopathogen	2.5	b
Repellent substance	3.27	b
Entomopathogen + Repellent	1.5	c
For each variable, different letters indicate differences according to LSD 5%.

Table 3: Postinfestation effects after treatment with an entomopathogen fungus and a repellent substance. Insect population inside the seeds. GFs were dissected at 15 days. * For each variable, differences with respect to the control (water) according to Dunnett's test at 5%. For each variable, different letters indicate differences according to LSD 5%.

Figure 6 shows the effect of a product with postinfestation effects, an entomopathogen, and that of a repellent substance, as well as their combined action.

Figure 6: Postinfestation effects of an entomopathogen fungus and a repellent substance. Percent mortality of adult H. hampei and seed damage. Please click here to view a larger version of this figure.

These methodologies allow the rapid determination of different effects of toxic products on the CBB.

Discussion

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 epidermis^7,15,16. 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 interfering with the bioassay results. Previously, Tapias et al.²⁰ evaluated other antimicrobial agents for fruit disinfection, such as carbendazim and benzalkonium chloride; however, although fruit disinfection was good, these compounds were highly toxic to the CBB or the environment.

The use of 0.5% sodium hypochlorite was evaluated by dipping the fruits in the solution for 30 min and 15 min. After both lengths of times, microorganisms were affected, but the CBB were also affected after 30 min of dipping due to the oxidizing power of the solution²³. UV light causes damage to microorganisms' DNA²⁴, decreasing contamination. However, at higher doses (longer exposure time), fruit damage occurs, causing necrosis and seed dehydration. Disinfection with 0.5% sodium hypochlorite for 15 min followed by UV light exposure for 15 min was found to be optimal in this procedure.

The second consideration is insect quality. For this study, the insects were provided by an insect rearing unit called BIOCAFE²⁵ (http://avispitas.blogspot.com/p/biocafe.html). Weak or inbred insects from poor insect colonies will overestimate the results of a toxic product. Moreover, the laboratory behavior, in this case, would not correspond to the field observations of wild-type insects with high fitness. Additionally, such insects can contain a large number of microorganisms that could interfere with the bioassay. Hence, disinfection²¹ is an important step to ensure the success of the methodology.

With respect to infestation (two insects to one fruit), it was previously determined that using a larger quantity of insects would increase the number of coffee fruits with more than one insect perforation, making the analysis more difficult²⁰. In addition, the temperature at which the experiments are conducted is important for obtaining fruits with insects in position A or obtaining a normal insect penetration when the fruits are sprayed. Using a temperature of 21 °C for 3 h allowed more than 70% of the fruits to be infested. When the temperature increased to between 25-27 °C, most of the insects reached position B in a shorter length of time than at 21 °C. The faster penetration of the CBB into the fruit is a consequence of the insect's greater activity due to temperature increase²⁶. Thus, the inconvenience of using a temperature of 25 °C for a longer period of time is that many fruits are found with more than one perforation, and with insects in both A and B positions.

Before the development of this method, artificial insect diets with ground coffee were used to evaluate the effects of toxic substances by incorporating the substance into or over the diet^5,6; however, these diets are expensive due to their special components^27,28. Parchment coffee has also been used for insecticide evaluation, where the coffee beans are sprinkled with or dipped into the substance to be evaluated. As the structure and composition of the parchment are different from those of the fruit's pericarp, it would be expected that the interaction between the insecticide and the coffee is different. With parchment coffee, the insecticide molecule can be easily absorbed, thus generating greater mortality than that observed under natural conditions. In addition, parchment coffee is comparatively more expensive since it has to be removed from the fruit pulp and then dried. Moreover, it is not the natural substrate for insect growth.

In conclusion, using real green coffee with nutrients adequate for insect growth is the most appropriate way to evaluate the toxicity of compounds to insects under simulated natural conditions.

Materials

Name	Company	Catalog Number	Comments
Beaker with spout, low form 500 mL	BRAND PP	BR87826
Benchtop Shaker	New Brunswick Scientific Innova 4000 Incubator Shaker
Dishwashing liquid soap-AXION	Colgate-Palmolive	AXION
Hood; Horizontal Laminar Flow Station	Terra Universal		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)
Insects CBB	BIOCAFE		(http://avispitas.blogspot.com/p/biocafe.html).
Multi Fold White paper towels	Familia	73551
Preval Spray unit	Preval Merck	Z365556-1KT	https://www.sigmaaldrich.com/CO/es/product/sigma/z365556?gclid=Cj0KCQiAweaNBhDEARIsAJ 5hwbfZOy1TWGj6huatFtRQt AzOyHe5-oBiKnOUK2T1exuuk WwJLdvxkvsaAjoYEALw_wcB
Reversible Racks 96-Well	heathrowscientific	HEA2345A	https://www.heathrowscientific.com/reversible-racks-96-well-i-hea2345a
Scalpel blades N 11	Merck	S2771-100EA
Scalpel handles N3	Merck	S2896-1EA
Sodium Hypochloride	The clorox company	Clorox
Stereo Microscope	Zeiss	Stemi 508	https://www.zeiss.com/microscopy/int/products/stereo-zoom-microscopes/stemi-508.html