HK40017674B - Compounds, compositions and methods for attracting and/or arresting bed bugs - Google Patents

Description

Compounds, compositions and methods for attracting and/or capturing bed bugs

This application is a divisional application. The application date of the original application is December 16, 2014, the application number is 201480069606.9, and the name of the invention is “Compounds, compositions and methods for attracting and/or capturing bed bugs”.

Field of the Invention

The present invention relates to compounds, compositions and methods for attracting and/or capturing bed bugs.

Background of the Invention

For many years, from World War II until the last decade of the 20th century, much of the world was free of infestations by the common bed bug, Cimex lectularius (Cimex lectularius) (Heteroptera: Cimicifuga). This was attributed to modern hygiene practices and the widespread use of DDT and other persistent insecticides (Potter 2012). However, in the last two decades, there has been a worldwide resurgence, with bed bugs becoming common urban pests (Boase 2001; Myles et al., 2003; Jones 2004) and sometimes causing debilitating skin irritation and lesions (Ter Porten et al., 2005). This resurgence has led to renewed interest in detecting, monitoring, and controlling bed bug infestations, specifically in the development of effective compounds, compositions, and methods for attracting and/or capturing bed bugs.

SUMMARY OF THE INVENTION

The invention described herein has several aspects.

In one aspect, a composition for attracting and/or capturing blood-feeding insects is provided. The composition includes histamine. The composition may further include volatile compounds, such as sulfides, aldehydes, and ketones, for example, dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone. The volatile compounds may include, by weight, 0.5-99% dimethyl disulfide, 0.5-99% dimethyl trisulfide, 0.5-99% (E)-2-hexenal, 0.599% (E)-2-octenal, and 0.5-99% 2-hexanone. The composition may further include an effective amount of one or more additional compounds, such as butyraldehyde, valeraldehyde, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenone, ethyl octanoate, methyl octanoate, amyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L-valine, L-alanine, octanal, nonanal, decanal, (E,E)-2,4-octadienal, (E,Z)-2,4-octadienal, benzyl acetate, (+)-limonene, ( -)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, capric acid, myristic acid, androstenone, 3-methylindole, 1-docosanol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol.

The histamine can be histamine base. The composition can be formulated into granules, powders, dusts, pastes, gels, suspensions, emulsions or liquid solutions. The composition can be formulated into a slow-release bait. The histamine component of the composition can be formulated into a slow-release bait. A blend of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone can be formulated into a slow-release bait. A blend of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone combined with an effective amount of one or more other compounds can be formulated into a slow-release bait. The one or more additional compounds may be selected from butyraldehyde, valeraldehyde, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenone, ethyl octanoate, methyl octanoate, amyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L-valine, L-alanine, octanal, nonanal, decanal, (E,E)-2,4-octadienal, (E,Z)-2,4-octadienal, benzyl acetate, (+)-limonene, (-)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, capric acid, myristic acid, androstenone, 3-methylindole, 1-docosanol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol.

In another aspect, a composition for attracting and/or capturing blood-feeding insects is provided, wherein the active ingredient of the composition consists essentially of histamine.

On the other hand, a composition for attracting and/or capturing blood-feeding insects is provided, wherein the active ingredients of the composition mainly consist of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone.

On the other hand, a composition for attracting and/or capturing blood-feeding insects is provided, wherein the active ingredients of the composition mainly consist of histamine, dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone.

In another aspect, a composition for attracting and/or capturing blood-feeding insects is provided, wherein the active ingredient of the composition consists essentially of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone in combination with an effective amount of one or more additional compounds selected from butyraldehyde, valeraldehyde, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenone, ethyl octanoate, methyl octanoate, amyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L-valine, L-alanine, octanal, nonanal, decane, octan ... aldehyde, (E,E)-2,4-octadienal, (E,Z)-2,4-octadienal, benzyl acetate, (+)-limonene, (-)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, capric acid, myristic acid, androstenone, 3-methylindole, 1-docosanol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol.

In another aspect, a composition for attracting and/or capturing blood-feeding insects is provided, wherein the active ingredient of the composition consists essentially of histamine, dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone in combination with an effective amount of one or more additional compounds selected from butyraldehyde, valeraldehyde, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenone, ethyl octanoate, methyl octanoate, amyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L-valine, L-alanine, octanal, nonanal, Decanal, (E,E)-2,4-octadienal, (E,Z)-2,4-octadienal, benzyl acetate, (+)-limonene, (-)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, capric acid, myristic acid, androstenone, 3-methylindole, 1-docosanol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol.

On the other hand, a method for attracting and/or catching blood-feeding insects is provided. The method comprises providing a composition described herein in a desired position. Histamine can be provided in a slow-release device comprising an absorbent material, such as a cellulose pad that is impregnated with histamine. Dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone can be provided in a slow-release device comprising a breathable sealed reservoir loaded with dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone. Dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone and one or more additional compounds can be provided in a sustained-release device comprising a gas-permeable sealed reservoir loaded with dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone and one or more additional compounds, wherein the one or more additional compounds are selected from butyraldehyde, valeraldehyde, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenone, ethyl octanoate, methyl octanoate, amyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L- The blood-feeding insect may be Cimex lectularius or C. hemipterus. The desired location may be in, on, or near a bed bug control device. The bed bug control device can be a detector, a monitor, or a trap. The slow-release device can be disposed in the detector, monitor, or trap. The method can further include combining the composition with a heat source. The method can further include combining the composition with a carbon dioxide source. The method can further include combining the composition with a bed bug lethal insecticide.

In another aspect, the invention provides a method for attracting and/or capturing blood-feeding insects. The blood-feeding insects may be the common bed bug Cimex lectularius or the tropical bed bug C. hemipterus.

In another aspect, the invention provides the use of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone for attracting and/or capturing blood-feeding insects. The blood-feeding insects may be the common bed bug Cimex lectularius or the tropical bed bug C. hemipterus.

In another aspect, there is provided the use of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone in combination with an effective amount of one or more additional compounds selected from butyraldehyde, valeraldehyde, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenone, ethyl octanoate, methyl octanoate, amyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L valine, L-alanine, octanal, nonanal, decanal, (E,E)-2-octenal, and 2-hexanone for attracting and/or capturing blood-feeding insects. ,4-octadienal, (E,Z)-2,4-octadienal, benzyl acetate, (+)-limonene, (-)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, capric acid, myristic acid, androstenone, 3-methylindole, 1-docosanol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol. The blood-feeding insect may be the common bed bug Cimex lectularius or the tropical bed bug C. hemipterus.

In another aspect, there is provided the use of histamine, dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone in combination with an effective amount of one or more additional compounds selected from butyraldehyde, valeraldehyde, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenone, ethyl octanoate, methyl octanoate, amyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L valine, L-alanine, octanal, nonanal, decanal, (E,E)- 2,4-octadienal, (E,Z)-2,4-octadienal, benzyl acetate, (+)-limonene, (-)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, capric acid, myristic acid, androstenone, 3-methylindole, 1-docosanol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol. The blood-feeding insect may be the common bed bug Cimex lectularius or the tropical bed bug C. hemipterus.

In another aspect, there is provided a use of the composition described herein for attracting and/or capturing blood-feeding insects. The blood-feeding insects may be the common bed bug Cimex lectularius or the tropical bed bug C. hemipterus.

The foregoing discussion merely outlines certain aspects of the invention and is not intended or should be construed as limiting the invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the design of (a) a three-dish dual-choice olfactometer and (b) a large Plexiglass platform; the inset illustrates a bed bug shelter composed of histamine-infused or control filter paper (FP), corrugated cardboard (CC), and an inverted bottle cap (IVL) containing either a volatile pheromone component formulated in mineral oil or mineral oil alone. The figures are not to scale.

Detailed description

Throughout the following description, the purpose of showing specific details is to provide a more comprehensive understanding of the present invention. However, the present invention can be practiced without these details. In other examples, well-known elements are not shown or described in detail to avoid unnecessary obscurity of the present invention. Therefore, the description will be considered as an example, not a limiting sense.

Some aspects of the present invention relate to compositions for attracting and/or capturing bed bugs (such as the common bed bug Cimex lectularius and the tropical bed bug C. hemipterus). In some embodiments, the composition may include a pheromone component isolated from the molted skin or feces of the bed bug Cimex lectularius, or a synthetic equivalent of such a compound. In some embodiments, the composition includes histamine. In some embodiments, the active ingredient of the composition may consist primarily of histamine. In some embodiments, the histamine is provided in base form. In some embodiments, the composition includes histamine and a blend of volatile compounds including sulfides, aldehydes, and ketones. In some embodiments, the blend of volatile compounds includes one or more of the following: dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone. In some embodiments, the active ingredient of the composition may consist primarily of histamine and one or more of the following: methyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone. In some embodiments, the composition can include or consist essentially of histamine, dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone.

In some embodiments, the compositions described herein may include one or more additional active ingredients, such as butyraldehyde, valeraldehyde, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenone, ethyl octanoate, methyl octanoate, amyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L-valine, L-alanine, octanal, nonanal, decanal, (E,E)-2,4-octadienal, (E,Z)-2,4-octadienal, benzyl acetate, esters, (+)-limonene, (-)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, capric acid, myristic acid, androstenone, 3-methylindole, 1-docosanol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol.

In some embodiments, the compositions described herein can be formulated as granules, powders, dusts, pastes, gels, suspensions, emulsions, or liquid solutions.

Certain aspects of the present invention relate to methods for attracting and/or capturing bed bugs (e.g., the common bed bug Cimexlectularius and the tropical bed bug C. hemipterus) at a desired location. In some embodiments, the methods comprise providing a composition described herein at a desired location. In some embodiments, the desired location can be in, on, or near a bed bug control device located in any human or animal dwelling, such as one or more living spaces in a residence, institution, commercial environment, industrial environment, or agricultural environment where bed bugs have been present, are present, or are suspected of being present. In some embodiments, suitable bed bug control devices include detectors, monitors, and traps.

In some embodiments, the composition can be formulated into a bait, such as a slow-release bait, and provided in, on, or near a bed bug control device. In some embodiments, the composition can be provided in a slow-release device that is provided in, on, or near a bed bug control device. In some embodiments, certain components of the composition can be provided in separate slow-release devices. For example, in some embodiments, the slow-release device for histamine can be an absorbent material impregnated with histamine (e.g., an absorbent pad, such as a cellulose pad), while the slow-release device for a volatile compound described herein or a volatile compound described herein and one or more additional compounds can be a breathable sealed reservoir (e.g., a sealed plastic reservoir that is permeable to pheromones). In some embodiments, the components of the composition can be provided in the same slow-release device.

In some embodiments, the compositions may be combined with a heat source, carbon dioxide, and/or a bed bug lethal insecticide.

Certain aspects of the present invention relate to the use of compounds and compositions for attracting and/or capturing bed bugs. In some embodiments, histamine is used to attract and/or capture bed bugs. In some embodiments, the compositions described herein are used to attract and/or capture bed bugs.

The present invention can be further understood by reference to the following examples, which are summarized and described in detail below.These examples are provided by way of illustration and are not intended to be limiting.

Overview of Examples

Bed bug cultures were maintained as a source of molts and feces and to supply insects for bioassays. Bioassays were conducted using a three-plate double-choice olfactometer and a large-platform olfactometer to determine the bioactivity of natural sources of pheromones, extracts from said sources, and synthetic chemicals identified from said extracts.

50, 10, 2 or 1 molts induced equivalent attraction and capture of ^3rd to ^5th instar bed bug nymphs in a two-choice still air olfactometer. In addition, molts stored at room temperature for two months remained as effective as molts freshly removed from a bed bug colony.

The methanol extract of 6 molts was bioactive at an equivalent dose and induced capture in a baited olfactometer, but the extracts of molts in hexane, ether, dichloromethane, and acetonitrile were ineffective in inducing capture of ^3rd to ^5th instar nymphs. These results suggest that this pheromone component has a highly polar molecular structure.

The pheromone components were separated for structural elucidation by sequential extraction of the molts in organic solvents of increasing polarity (hexane, ether, dichloromethane, acetonitrile, and methanol) such that the final methanol extract contained the major polar compounds. The methanol extract was then fractionated through silica gel in a glass column by elution with five consecutive washes (2 ml each) of pentane/ether with increasing ether ratios [1) 100:0; 2) 90:10; 3) 80:20; 4) 50:50; 5) 0:100], followed by five consecutive washes (1 ml each) of dichloromethane/methanol with increasing methanol ratios [1) 100:0; 2) 90:10; 3) 80:20; 4) 50:50; 5) 0:100]. Bioassays with five dichloromethane/methanol fractions showed that only the fractions with dichloromethane (CH ₂ Cl ₂ ; 50%) and methanol (MeOH; 50%) as eluents induced a capture response in 3 ^rd to 5 ^th instar bed bug nymphs.

Methanol extracts of bed bug molts were subjected to microanalysis using diazomethane or acetic anhydride in pyridine, followed by bioassays. Biological activity was retained after diazomethane treatment but not after acetic anhydride treatment. These experiments indicate that key pheromone components possess one or more hydroxyl and/or amino groups.

The molecular structures of potential aggregation pheromone components were elucidated by subjecting the bioactive silica fraction containing only a few components to high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis using acetonitrile as the eluent, and one- and two-dimensional nuclear magnetic resonance (NMR) experiments. The results of these analyses revealed several components identified as N-acetylglucosamine, L-3-hydroxykynurene O-sulfate, L-valine, L-alanine, histamine, and dimethylaminoethanol.

A mixture of histamine and dimethylaminoethanol captured both sexes and all developmental stages of bed bugs in a three-dish two-choice olfactometer, but the effect of dimethylaminoethanol was uncertain. N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L-valine, and L-alanine were determined not to be essential pheromone components.

Headspace volatiles from filter paper contaminated with bed bug feces were analyzed using an Agilent headspace analyzer coupled to a Varian 2000 ion trap GC-MS equipped with a DB-5MS GC column (30 m x 0.25 μm ID). The analysis revealed a blend of 15 oxygenated or sulfur-containing volatile components consisting of six aldehydes (butyraldehyde, valeraldehyde, hexanal, (E)-2-hexenal, (E)-2-octenal, benzaldehyde), one alcohol (benzyl alcohol), three ketones (2-hexanone, acetophenone, verbenone), three esters (methyl octanoate, ethyl octanoate, amyl hexanoate), and two sulfides (dimethyl disulfide, dimethyl trisulfide). Doses of 50, 5.0, and 0.5 μg of the resulting blend of all 15 volatiles were used to capture bed bugs in a three-dish, double-choice olfactometer.

A series of subtraction experiments testing the 15-component synthetic blend individually or in key component groups in a three-dish double-choice olfactometer indicated that one or more of aldehydes, sulfides, and ketones or alcohols were essential bed bug pheromone components. Further subtraction experiments determined that the essential attractive volatile pheromone components for bed bugs were among the following six compounds: (E)-2-hexenal, (E)-2-octenal, dimethyl disulfide, dimethyl trisulfide, acetophenone, and 2-hexanone. A final series of subtraction experiments determined that acetophenone was not essential and that both sulfides and both aldehydes contributed to the biological activity of the blend. Therefore, it was concluded that the essential attractive volatile pheromone components were (E)-2-hexenal, (E)-2-octenal, dimethyl disulfide, dimethyl trisulfide, and 2-hexanone.

Bioassays in a large platform olfactometer with shelter traps at either end revealed that the highest captures were obtained when the traps were baited with low volatility histamine and a five-component volatile composition consisting of (E)-2-hexenal, (E)-2-octenal, dimethyl disulfide, dimethyl trisulfide, and 2-hexanone, and that the low volatility and volatile pheromone components acted synergistically.

Field experiments in which baited or control bunker traps were established in infested homes confirmed the synergistic interaction between low-volatility histamine and a five-component volatile composition consisting of (E)-2-hexenal, (E)-2-octenal, dimethyl disulfide, dimethyl trisulfide, and 2-hexanone, indicating that all six synthetic pheromone components are necessary for manipulated bed bug trapping. Field experiments also demonstrated that bunker traps baited with the six-component composition were highly effective in revealing bed bug infestations in homes with very low levels of bed bug infestation.

Example 1

Rearing colonies of common bed bugs to generate molts and for bioassays

A common bed bug colony was maintained in an insectary at 22-24°C, ambient relative humidity, and a photoperiod of 10 hours dark to 14 hours light. The colony was increased from 2,400 to 6,000 bed bugs and maintained at a high level for 18 months to collect pheromones for extraction, isolation, and identification.

Approximately 150 bed bugs were housed in 40 50-ml jars. Each jar was fitted with a piece of cardboard (2 x 2 cm) at the bottom and a corrugated cardboard strip (2 x 5 cm) running diagonally across the jar. The jar was covered with a plastic lid perforated with ventilation holes.

Each bed bug was allowed to be fed once a month on a human volunteer. 1,500 bed bugs per week for 30 months equates to 180,000 individual feedings. The lid of the jar with the bed bugs to be fed had a fine mesh and was pressed against the volunteer's forearm, allowing the bed bugs to feed through the mesh. After feeding, the nymphal bed bugs molted, shedding their exuviae in the process. Each exuviae of a 5th instar nymph weighed approximately 0.07 mg. Exuviae from 1,200 5th instar nymphs (20% of the entire population) were collected each month, resulting in a harvest of 84 mg (1200 x 0.07 mg) of exuviae per month, for a total of 1,512 mg of exuviae. This was the starting material for the extraction, isolation, and characterization of the aggregation pheromone.

Example 2

Overall Experimental Design to Examine Bed Bug Responses to Test Stimuli

Bioassays were conducted in a double-choice olfactometer (Figure 1) within a large Plexiglass platform. The double-choice olfactometer consists of two flanking glass Petri dishes connected to a central dish (all dishes were 3 x 9 cm inner diameter) via glass tubing (2.5 cm long x 2 cm inner diameter). The dishes in this olfactometer simulate the natural still-air shelters in which bed bugs spend their days. Before the bioassay began, a paper towel disk (9 cm diameter) was placed in each dish, and a paper towel strip (2.4 x 0.6 cm) was inserted into the connecting glass tubing to provide traction for the walking bed bugs. Additionally, a filter paper disc (2 x 3 cm; Whatman) was placed in each flanking dish and covered with a cardboard disc (2 x 2 cm) to serve as a shelter for the bioassay insects.

Treatment and control stimuli were randomly assigned to each flank culture dish. The olfactometer was enclosed in an opaque plastic box to prevent light from entering and affecting the insect's response. For each replication, a single ^3rd , ^4th , or ^5th instar C. lectularius nymph was released into the central chamber of the olfactometer, which was then covered with a glass plate to prevent escape. A single bed bug in each olfactometer was then allowed to visit all chambers. Each insect was released into the olfactometer at the end of the 14-h light period and allowed to visit the chamber during the 10-h dark period and rest in one of the shelters during the 10-h light period. After this 20-h period, the insect's position within the olfactometer was recorded. Any insect not found in the flank chamber was recorded as a non-responder. All experimental replications were carried out at 24 ± 2°C and 30-60% RH. Between each bioassay, the olfactometers were washed with Sparkleen detergent (Fisher Scientific Co, Pittsburgh, PA, USA) and oven-dried (Precision, Winchester, VA, USA).

A large two-chamber Plexiglass platform (180 cm long x 12 cm high x 13 cm wide) was designed with a central divider (180 cm long x 5.5 cm high) to accommodate two pieces of wood (162 cm long x 3.8 cm wide x 0.8 cm high), each of which was used to test the response of a single bed bug to either a pheromone-baited shelter or a control shelter (see inset in Figure 1b), which was randomly assigned to either end of the wood. As some bed bugs successfully crossed the divider from one chamber to the other, only one chamber of each platform was used for all experimental replicates. For each replicate, a single bed bug was placed in the center of the wood piece just before the end of the 14-hour light period, and its position was recorded the next morning after the start of the light painting.

Example 3

Demonstrating that bed bug molts (shedding of the outer skin during molting) induce capture of foraging bed bugs: Testing the effects of number of molts and aging

Experiment 1 tested whether 50 molts (1 molt = 0.08 mg) from ^5th instar bed bug nymphs induced capture of foraging ^5th instar nymphs. If nymphs responded strongly to capture of 50 molts (see Table 1), subsequent Experiments 2-4 tested whether smaller numbers of molts were sufficient to induce a capture response. Experiment 5 explored whether molts aged for 2 months at room temperature were still effective in inducing capture of bed bugs.

In Experiments 1-4, 50, 10, 2 or 1 molt were unexpectedly all equally effective in inducing bed bug capture (Table 1). Surprisingly, in Experiment 5, molts after 2 months of storage at room temperature still induced bed bug capture.

Example 4

Effectiveness of organic solvents in extracting pheromones from molting skin

Experiments 6-10 tested the effectiveness of different organic solvents (hexane, ether, dichloromethane, acetonitrile, methanol) in extracting pheromones from molts. Each olfactometer experiment tested six molt equivalents of extract, i.e., the amount of material potentially including pheromones that could be extracted from a total of six molts.

In Experiments 6-9, hexane, ether, dichloromethane, and acetonitrile were not effective in extracting pheromones from molts and inducing capture responses in ^3rd to ^5th instar nymphs (Table 2). In Experiment 10, the methanol extract of the molts induced a strong capture response (Table 2), indicating that the bed bug capture pheromone components were present in the methanol extract.

Example 5

Separation of capture pheromone components

The presence of capture pheromone components in the methanol extract of molts (Table 2, Experiment 10) suggests that they possess a predominantly polar molecular structure. To isolate the pheromone components for structural elucidation, molts were sequentially extracted in organic solvents of increasing polarity (hexane, ether, dichloromethane, acetonitrile, and methanol). The final methanol extract contained predominantly polar compounds. This methanol extract was then fractionated through silica gel (0.6 g) in a glass column (14 cm long x 0.5 cm inner diameter). After the silica gel was pre-rinsed with pentane, the methanol extract was applied, allowed to perfuse the silica gel, and then eluted using five consecutive washes of pentane/ether (2 ml each) with increasing ether ratios [1) 100:0; 2) 90:10; 3) 80:20; 4) 50:50; 5) 0:100], followed by five consecutive washes of dichloromethane/methanol (1 ml each) with increasing methanol ratios [1) 100:0; 2) 90:10; 3) 80:20; 4) 50:50; 5) 0:100]. The five dichloromethane/methanol fractions were then bioassayed in experiments 11-15.

In Experiments 11-15 (Table 3), only the silica fraction with 50% methanol as the eluent (Experiment 14) induced capture of bed bug nymphs.

This pheromone isolation protocol was repeated several times in the same or slightly modified form. Bioassays of insect responses to the silica fractions each time revealed that the capture pheromone component was present in the fractions eluted with either 50% or 100% methanol. These combined results clearly revealed that the capture pheromone component was highly polar.

Example 6

Pheromone Identification: Microanalytical Processing of Bioactive Extracts

To determine whether the polar capture pheromone components possessed acid, amine, or alcohol functionality, methanol extracts of molts (see Table 2) were subjected to microanalysis using either diazomethane (to convert acids to esters) or acetic anhydride in pyridine (to convert alcohols to esters) and then bioassayed in Experiments 16 and 17.

In Experiment 16 (Table 4), the methanol extract of the diazomethane-treated molts induced a capture response in bed bug nymphs, indicating that the diazomethane treatment did not alter the pheromone molecule and that this pheromone component is unlikely to have acid functionality. In contrast, in Experiment 17, the methanol extract of the molts treated with acetic anhydride did not induce capture in bed bug nymphs, indicating that the acetic anhydride treatment had altered the molecular structure of the pheromone component and that it possessed one or more hydroxyl and/or amino groups.

Example 7

Pheromone identification: Nuclear magnetic resonance spectroscopy (NMR) of bioactive extracts

To elucidate the molecular structure of the pheromone components, ^1H NMR spectra of several methanol extracts of molts and feces were examined and compared. ^1H NMR spectral analysis revealed several common components, which were identified as L-valine, L-alanine, N-acetylglucosamine, histamine, dimethylaminoethanol, and 3-hydroxykynurenine O-sulfate.

Valine, alanine, N-acetylglucosamine, histamine, and dimethylaminoethanol were identified by comparing the observed ¹ H and ¹³ C NMR and mass spectral data for these compounds with those previously reported. Additionally, certified samples of L-valine, N-acetylglucosamine, histamine, and dimethylaminoethanol were purchased from suppliers and spiked into the crude methanol extract in separate experiments. In each of these additional experiments, resonances observed in the ¹ H NMR spectrum recorded for the crude methanol extract and assigned to specific components (i.e., L-valine, L-alanine, N-acetylglucosamine, histamine, and dimethylaminoethanol) were enhanced by the addition of certified samples of these components, verifying the identity of these chemicals and their presence in the crude methanol extract.

The structure of 3-hydroxykynurenine O-sulfate was proposed based on a comparison of specific resonances observed in the ^1H NMR spectrum recorded for the crude methanol extract with those previously reported for 3-hydroxykynurenine O-sulfate. Additionally, a certified sample of 3-hydroxykynurenine O-sulfate was prepared from 3-hydroxykynurenine by protecting the carboxylic acid function with a methyl ester and the amine function with a carboxybenzylamide. Sulfation of the free alcohol using _Me₃N — _SO₃ followed by removal of the carboxybenzyl protecting group by hydrogenolysis and hydrolysis of the methyl ester provided a certified sample of 3-hydroxykynurenine O-sulfate. The 1D and 2D NMR spectra ( ^1H , COSY, HMQC, HMBC) recorded for this synthetic sample of 3-hydroxykynurenine O-sulfate were fully consistent with those of the natural material present in the crude methanol extract.

Table 5 summarizes the relative amounts of each of these components in each of the five individual methanol extracts, and Table 6 shows the biological activity of each extract from Experiments 18 to 22. Note that the three extracts in which both histamine and dimethylaminoethanol were present in appreciable amounts achieved the highest response levels (Experiments 18 to 20).

Example 8

Histamine: An essential pheromone component for bed bugs

Since extracts containing both histamine and dimethylaminoethanol achieved the highest response levels in bed bugs (Tables 5 and 6), subsequent experiments were designed to test bed bug responses to certified standards. In Experiments 23-25, doses of dimethylaminoethanol and histamine in a 1:1 ratio increased bed bug capture responses (Table 7). Experiments 26-28 then tested dimethylaminoethanol (20 μg) and histamine (20 μg), with histamine present as a base, a salt, or both. The results revealed that only histamine, as a base, elicited a bed bug capture response (Table 7). Therefore, all further experiments utilized histamine as a base.

Experiments 29-31 tested whether a 10-fold increase (from 20 μg to 200 μg) in dimethylaminoethanol or histamine improved bait effectiveness. Results showed that a 10-fold increase in histamine enhanced the bed bug capture response (Table 7). Leveraging the results of Experiments 29-31, Experiments 32-34 tested dimethylaminoethanol and histamine at a 1:10 ratio of 2 μg:20 μg, 20 μg:200 μg, and 200 μg:2000 μg. Results showed that the two highest doses were highly effective in eliciting a bed bug capture response (Table 7). To determine the effects of dimethylaminoethanol or histamine in a two-component blend, Experiments 35-37 tested 200 μg each of dimethylaminoethanol and histamine alone and in a 20 μg:200 μg ratio. The data revealed that histamine was biologically active on its own, while dimethylaminoethanol was not, and was equally effective at capturing bed bugs as the two-component blend at the doses tested.

Experiments 23–37 failed to identify a clear effect of dimethylaminoethanol. On the one hand, when dimethylaminoethanol and histamine were provided in a 2 μg:20 μg ratio in Experiment 32, there was no preference for the feeding chamber. However, when the dimethylaminoethanol dose was increased to 20 μg, equal to the histamine dose in Experiments 24 and 29, a preference for the feeding chamber emerged. On the other hand, unlike histamine in Experiment 37, dimethylaminoethanol alone was inactive in Experiment 36. Furthermore, in Experiment 32, a 10-fold increase in the dimethylaminoethanol dose did not result in increased responding when the histamine dose remained constant.

Example 9

Effects of Additional Components (L-Valine, L-Alanine, N-Acetyl Glucosamine, 3-Hydroxy-Kynurenic Acid O-Sulfate) on Histamine and Dimethylaminoethanol Blends

Experiments 38 and 39, and 40 and 41 tested whether a two-component blend of dimethylaminoethanol and histamine (20 μg:200 μg) would be more effective by adding L-valine, L-alanine, and N-acetylglucosamine (Experiment 39) or 3-hydroxy-kynurenine O-sulfate (Experiment 41) when other components were present in methanol extracts of bed bug molts or feces (Table 5). The data revealed that the effectiveness of the two-component blend of dimethylaminoethanol and histamine in capturing bed bugs (Experiments 38 and 40) could not be improved by the addition of other components (Table 8).

Example 10

Comparative responses of bed bug nymphs, adult males, and adult females to a mixture of dimethylaminoethanol and histamine

To determine whether a two-component blend of dimethylaminoethanol and histamine elicited a capture response in both immature and mature stages of bed bugs, experiments 42-44 tested the responses of bed bug nymphs, adult males, and adult females to dimethylaminoethanol and histamine (20 μg: 200 μg). The data revealed that the two-component blend was equally effective in inducing a capture response in nymphs and adult males and females (Table 9).

Example 11

Identification of candidate volatile pheromone components in bed bug feces

Given that dimethylaminoethanol has only limited pheromone effects (Table 7), and that histamine is a low volatility compound and therefore likely acts as a trapping agent rather than an attractant, the search for attractive volatile pheromone components continued. The focus shifted to bed bug feces present along with molting in natural bed bug shelters.

Filter paper sheets (5 x 10 cm) exposed to the feces of approximately 300 bed bugs over a four-week period were cut into 0.75 x 0.5 cm sections and analyzed using an Agilent headspace analyzer coupled to a Varian 2000 ion trap GC-MS equipped with a DB-5MS GC column (30 m x 0.25 μm ID). After placing the fecal-stained paper in a 20-ml vial, the vial was sealed with a crimp cap fitted with a 20-mm OD white silicon septum and heated to 90°C for 5 minutes. Onboard headspace volatiles were extracted using an automated syringe and analyzed by coupled gas phase GC-MS using the following temperature program: 50°C for 5 minutes, then 10°C per minute to 280°C. The analysis revealed a complex mixture of 15 oxygenated or sulfur-containing volatile components consisting of six aldehydes (butyraldehyde, valeraldehyde, hexanal, (E)-2-hexenal, (E)-2-octenal, benzaldehyde), one alcohol (benzyl alcohol), three ketones (2-hexanone, acetophenone, verbenone), three esters (methyl octanoate, ethyl octanoate, amyl hexanoate), and two sulfides (dimethyl disulfide, dimethyl trisulfide). All 15 compounds were considered candidate pheromone components and tested for bed bug attraction in a two-choice olfactometer bioassay (see Example 2; Figure 1).

Example 12

Responses of adult male bed bugs to three doses of a synthetic blend of bed bug fecal volatiles

To determine whether a blend of 15 bed bug fecal volatiles identified in bed bug feces (see Example 11) attracts bed bugs and therefore contains one or more bed bug pheromone components, bed bug responses to a synthetic blend (SB) at medium (50 μg; Experiment 45), low (5 μg; Experiment 46), and very low (0.5 μg; Experiment 47) doses were tested in a three-dish, two-choice olfactometer (see Example 2, Figure 1). At each dose tested, all components were prepared in equal amounts in mineral oil and pipetted into the inverted caps of 4-ml vials. The vials were then placed on top of corrugated cardboard covers in randomly assigned treatment dishes in the olfactometer (see Figure 1) (see Example 2). A control stimulus consisted of the same type of cover—containing mineral oil without the synthetic test chemical—on the corrugated cardboard of the control dish in the olfactometer.

In experiments 45-47, the synthetic blends of bed bug fecal volatiles at medium, low, and very low doses all attracted more male bed bugs than the control stimulus, indicating that the blends contained one or more bed bug pheromone components. The medium dose was selected for further experiments.

Example 13

Determination of pheromone components in synthetic blends of bed bug fecal volatiles

To determine the essential component(s) of a 15-component synthetic blend of bed bug fecal volatiles (SB) that attracts bed bugs (Example 12; Table 10), Experiments 48-52 compared responses to the full 15-component intact SB (Experiment 48) with SB lacking a relevant group of organic chemicals (i.e., esters (methyl octanoate, ethyl octanoate, amyl hexanoate; Experiment 49), aldehydes (butyraldehyde, valeraldehyde, hexanal, E2-hexenal, E2-octenal, benzaldehyde; Experiment 50), sulfides (dimethyl disulfide, dimethyl trisulfide; Experiment 51), and ketones/alcohols (2-hexanone, acetophenone, verbenone, benzyl alcohol; Experiment 52). All SBs were formulated and bioassayed as described in Example 11.

In Experiment 48 (Table 11), 15-component SB strongly attracted adult male bed bugs, confirming that this SB contains one or more bed bug pheromone components. Similarly, in Experiment 49 (Table 11), SB lacking esters strongly attracted adult male bed bugs, indicating that esters are not an essential part of the bed bug pheromone blend. In contrast, shelters baited with SB lacking aldehydes (Experiment 50), sulfides (Experiment 51), or ketones and alcohols (Experiment 52) did not capture significantly more adult male bed bugs than control shelters, indicating that aldehydes, sulfides, and one or more of ketones or alcohols are essential bed bug pheromone components.

To narrow down the specific aldehyde(s), ketone(s), or sulfide(s) that constitute the components of the bed bug pheromone, subsequent and concurrent experiments again tested intact SB (Experiment 53), and SB lacking benzaldehyde and benzyl alcohol (Experiment 54), verbenone (Experiment 55), or both (E)-2-hexenal and (E)-2-octenal (Experiment 56).

In Experiment 53 (Table 11), shelters baited with 15-component SB captured twice as many adult male bed bugs as control shelters. Similar data were obtained with SB lacking benzaldehyde and benzyl alcohol (Experiment 54) or verbenone (Experiment 55), indicating that none of these three compounds are essential components of the bed bug pheromone. In contrast, shelters baited with SB lacking both (E)-2-hexenal and (E)-2-octenal (Experiment 56) did not capture more adult male bed bugs than control shelters, indicating that one or both of these aldehydes are components of the bed bug pheromone.

The combined data from Experiments 48-56 indicated that the essential pheromone components for bed bug attraction were among the following six compounds: (E)-2-hexenal, (E)-2-octenal, dimethyl disulfide, dimethyl trisulfide, acetophenone, and 2-hexanone. To determine whether this six-component synthetic blend (6-component SB) was as effective as 15-component SB in attracting bed bugs, Experiments 57 and 58 tested SB and 6-component SB against a control stimulus.

In Experiment 57 (Table 11), the 15-component SB attracted significantly more adult male bed bugs than the control stimulus, as expected. In Experiment 58, the 6-component SB attracted significantly more adult male bed bugs than the control stimulus. Since SB and the 6-component SB exhibited equivalent ability to attract bed bugs, it was concluded that all necessary pheromone components were present in the 6-component SB.

To determine the essential ketone(s) in 6-component SB, subsequent and concurrent experiments compared responses to 6-component SB (Experiment 59), 6-component SB lacking acetophenone (Experiment 60), and 6-component SB lacking 2-hexanone (Experiment 61).

In Experiment 59 (Table 11), 6-component SB attracted significantly more adult male bed bugs than the control stimulus. Similarly, in Experiment 60, 6-component SB lacking acetophenone attracted significantly more adult male bed bugs than the control stimulus, indicating that acetophenone is not an essential bed bug pheromone component. In contrast, in Experiment 61, 6-component SB lacking 2-hexanone failed to attract a significant number of adult male bed bugs, indicating that 2-hexanone is a key bed bug pheromone component.

To identify the key sulfide(s) in 6-component SB, the next three subsequent and concurrent experiments compared the responses to 6-component SB (Experiment 62), 6-component SB lacking dimethyl disulfide (Experiment 63), and 6-component SB lacking dimethyl trisulfide (Experiment 64).

In Experiment 62, the 6-component SB attracted significantly more adult male bed bugs than the control stimulus. Similarly, the two 6-component SBs lacking dimethyl disulfide (Experiment 63) or dimethyl trisulfide (Experiment 64) were still more effective at attracting adult male bed bugs than the control stimulus. However, the 6-component SB with both sulfides (Experiment 62) exhibited relatively higher attractiveness than the SB containing only one sulfide (Experiments 63 and 64), indicating that both sulfides are important pheromone components and should be included in manipulative baits.

To identify the key aldehyde(s) in 6-component SB, the next three subsequent and concurrent experiments compared responses to 6-component SB (Experiment 65), 6-component SB lacking (E)-2-hexenal (Experiment 66), and 6-component SB lacking (E)-2-octenal (Experiment 67).

In Experiment 65 (Table 11), the 6-component SB attracted significantly more adult male bed bugs than the control stimulus. Similarly, in Experiment 66, the 6-component SB lacking (E)-2-hexenal attracted significantly more adult male bed bugs than the control stimulus. In contrast, in Experiment 67, the 6-component SB lacking (E)-2-octenal attracted only twice as many adult male bed bugs as the control stimulus. The combined data reveal that (E)-2-octenal is a relatively more important pheromone component than (E)-2-hexenal. However, both aldehydes should be included in commercial bed bug pheromone baits for optimal attraction.

Example 14

Responses of bed bugs to synthetic pheromone baits in a large-scale bioassay platform

Experiments 68-75 were conducted to determine whether bed bugs respond to synthetic pheromones not only in a small olfactometer (Example 2; Figure 1; Tables 7-11), but also in a large bioassay platform. Experiments 68-75 also examined the effects of the volatile pheromone components (VPCs) (E)-2-hexenal, (E)-2-octenal, dimethyl disulfide, dimethyl trisulfide, and 2-hexanone, as well as the low volatility pheromone component histamine (H), on bed bug attraction and capture. Treatment stimuli consisted of filter paper (4 x 2.2 cm) impregnated with H (2,000 μg) and covered with a corrugated cardboard sheet shelter (3 x 2.2 cm); and VPC, either high dose (500 μg; Experiments 68-74) or medium dose (50 μg; Experiment 75) prepared in mineral oil (0.5 ml) and pipetted into the inverted cap of a 20-ml scintillation vial placed above the shelter ( FIG1 ). Control stimuli had the same design, but the filter paper did not contain H, and the mineral oil did not contain VPC.

Specifically, Experiment 68 tested a complete synthetic pheromone blend consisting of VPC and H against a control stimulus, while the concurrent Experiment 69 tested a partial pheromone blend consisting of VPC alone against a control stimulus.

In Experiment 68 (Table 12), 15 of the 16 male bed bugs tested each responded to the complete pheromone blend, indicating that the complete blend contains all the essential bed bug pheromone components. In Experiment 69, in contrast, only 6 of the 16 male bed bugs tested responded to the partial pheromone blend consisting of VPC without histamine, confirming that histamine is an essential bed bug pheromone component (see also Tables 7-9) and that histamine plays a role in capturing bed bugs in the shelter after they are attracted to the shelter by VPC.

To determine the effect of VPC in the pheromone blend, histamine alone was then tested against a vehicle control (Experiment 70), as was a complete pheromone blend of VPC and histamine against a vehicle control (Experiment 71).

In Experiment 70 (Table 12), 12 bed bugs responded to the shelter with the histamine-impregnated filter paper, while only one bed bug responded to the shelter with the blank filter paper, again indicating that bed bugs were captured in the presence of histamine. In Experiment 71, 17 bed bugs responded to the shelter associated with the complete pheromone blend of VPC and histamine, while only one bed bug responded to the control shelter, indicating that the complete pheromone blend may be more effective in attracting or capturing bed bugs than the partial blend (tested in Experiment 70).

To further compare the relative importance of histamine and VPC as components of the bed bug pheromone, bed bug responses were tested simultaneously to shelters baited with either histamine or VPC (Experiment 72) and a complete pheromone blend of VPC and histamine versus a blank control (Experiment 73).

In Experiment 72 (Table 12), 14 bed bugs responded to the shelter baited with histamine, while 2 responded to the shelter baited with VPC, indicating that histamine had a stronger influence on the bed bugs' decision to choose a shelter. In Experiment 73, 15 bed bugs responded to the shelter baited with the complete pheromone blend (VPC + histamine), while only 1 bed bug responded to the unbaited control shelter, confirming the superior effect of the complete bed bug pheromone blend.

Given that histamine strongly captures bed bugs in shelters (see experiments 70 & 72 in Table 12), we then explored whether the effects of histamine could be enhanced by adding VPC to histamine. Therefore, we tested the response of bed bugs to histamine or to histamine + VPC, both at a high dose of VPC (500 μg; experiment 75) and a medium dose of VPC (50 μg; experiment 74).

In Experiments 74 and 75, the complete pheromone blends of histamine plus high and medium doses of VPC attracted and captured significantly more bed bugs than histamine alone, clearly revealing a synergistic effect between VPC (attracting bed bugs) and histamine (capturing bed bugs).

Example 14

Response of bed bugs to synthetic pheromone baits in heavily infested homes

To determine whether bed bugs respond to synthetic pheromone baits not only in large-scale laboratory bioassays but also in infested homes (houses), pheromone-baited shelter traps (see Figure 1) were deployed in homes known or suspected to be infested with bed bugs. The shelters were identical to those described in Example 13, except that histamine-infused filter paper or control filter paper was glued to corrugated cardboard shelters to facilitate shelter deployment. Based on the 24-hour trapping results in multiple homes, a single heavily infested home (hereinafter referred to as Room 106) was selected for further testing with synthetic bed bug pheromone baits.

To obtain field evidence for a synergistic interaction between VPC and histamine in synthetic pheromone baits (see laboratory experiments 74 & 75 in Table 12), two consecutive two-experimental groups were conducted in chamber 106 (Group 1: Experiments 76 and 77; Group 2: Experiments 78 and 79). Each experimental replicate (n = 15-20) consisted of pairs of shelters arranged against a wall or furniture, with approximately 30 cm spacing between pairs and > 1 m spacing between shelter pairs. Pheromone and control treatments were randomly assigned to each shelter pair within each experiment, and the replicate placement was alternating between the two experiments in Groups 1 and 2. Because chamber 106 could accommodate no more than 10 shelter pairs at any one time, experimental data were collected over several consecutive 24-hour periods. Each day at 9:00 AM, shelters were collected from chamber 106 and immediately placed in individual labeled zip-lock bags, which were then placed on dry ice to kill any bed bugs that had entered the shelters. The bed bugs in each shelter were then counted under a microscope in the laboratory and annotated with their developmental stage ( ^1st , ^2nd , ^3rd , ^4th instar, adult), sex (male, female), and evidence of blood feeding before entering the shelter (or none). Each morning, immediately after the bed bug shelters were removed from chamber 106, new, prepared shelters were either baited with pheromones (treated) or unbaited (control) and randomly assigned to their positions within the shelter pairs (see above), again alternating between Experiments 76 and 77 (Group 1) and Experiments 78 and 79 (Group 2).

To determine the effectiveness of synthetic pheromone baits in the presence or absence of the volatile pheromone components (VPCs) (E)-2-hexenal, (E)-2-octenal, dimethyl disulfide, dimethyl trisulfide, and 2-hexanone (predicted to attract bed bugs to shelters) and the low volatility component histamine (predicted to retain bed bugs in shelters), bed bug responses to complete and partial synthetic pheromone baits were compared in Experiments 76 and 77 and in Experiments 78 and 79, each set of experiments being conducted simultaneously.

In Experiment 76, shelters baited with a complete synthetic pheromone bait (VPC + histamine) were tested versus unbaited shelters, and in Experiment 77, shelters baited with VPC alone were tested versus unbaited shelters. Similarly, in Experiment 78, shelters baited with a complete synthetic pheromone bait (VPC + histamine) were tested versus unbaited shelters, and in Experiment 79, shelters baited with histamine alone were tested versus unbaited shelters.

In Experiment 76 (Table 13), shelters baited with the complete synthetic pheromone blend attracted and retained an average of 21.4 bed bugs, compared to an average of 4.8 bed bugs in the control shelters. These data indicate that the complete synthetic pheromone bait has a significant effect on attracting and retaining bed bugs. In contrast, in Experiment 77, shelters baited with VPC alone attracted and retained an average of only 10.3 bed bugs, compared to an average of 4.7 bed bugs in the control shelters.

The combined data from Experiments 76 and 77 revealed that the complete synthetic pheromone bait was superior to the partial pheromone bait (lacking histamine) in attracting and retaining bed bugs.

In Experiment 78 (Table 13), shelters baited with the complete synthetic pheromone blend attracted and retained an average of 24.86 bed bugs, compared to an average of 3.73 bed bugs in the control shelters. These data again demonstrate that the complete synthetic pheromone bait is significantly effective in attracting and retaining bed bugs. In contrast, in Experiment 79, shelters baited with the partial pheromone blend containing only histamine attracted and retained an average of only 6.06 bed bugs, compared to an average of 4.53 bed bugs in the control shelters.

The combined data from Experiments 76-79 revealed that the complete synthetic pheromone blend was the most effective at attracting and retaining bed bugs. The surprisingly superior performance of the complete synthetic pheromone blend stems from the combined effects of two types of pheromone components: volatile components (VPCs) that attract bed bugs to the shelter, and myramine, a low-volatility pheromone component that traps the bed bugs in the shelter after they are attracted.

Pheromone-baited shelters contained all nymphal instars (fed and unfed) as well as adult males and females (fed and unfed), clearly demonstrating that the intact synthetic pheromone attracted and retained bed bugs regardless of their developmental stage, sex, or physiological state.

Example 15

Response of bed bugs to synthetic pheromone baits in lightly infested homes

To determine whether bed bugs respond to whole synthetic pheromones not only in heavily infested homes (see Example 14; Table 13) but also in homes with minimal infestations, trapping was conducted in eight homes in three separate buildings where occupants suspected bed bugs based on reports from pest management professionals. In each of these homes, bedding, upholstery, and other furniture were carefully inspected for the presence of live bed bugs and evidence of bed bug activity, such as fecal spots on bedsheets. In Experiment 80, 1-5 pairs of bed bug shelter traps (see Figure 1) were placed in each home, for a total of 20 pairs. One shelter in each pair was baited with the pheromone [VPC: (E)-2-hexenal, (E)-2-octenal, dimethyl disulfide, dimethyl trisulfide, 2-hexanone (500 μg) + histamine (2,000 μg)]; the other was unbaited. Shelter placement and spacing within and between shelter pairs were as described in Example 14. The shelters were retrieved 24 hours after deployment, immediately placed in individually labeled zip lock bags, and placed on dry ice to kill any captured bed bugs.

In Experiment 80, 26 of the 27 bed bugs captured were found in pheromone-baited shelters; only one bed bug was found in the unbaited control shelter. All shelter traps in chambers where at least one live bed bug was observed at the start of the experiment captured at least one bed bug.

These data indicate that a six-component synthetic pheromone bait consisting of (E)-2-hexenal, (E)-2-octenal, dimethyl disulfide, dimethyl trisulfide, 2-hexanone, and histamine can attract and retain bed bugs in lightly infested homes and that this novel pheromone bait has the potential to be an effective means of detecting bed bug infestations in residential and commercial buildings.

This application is intended to cover any adaptation, application, or modification of the present invention that utilizes the general principles of the present invention. Furthermore, this application is intended to cover modifications of the present disclosure that come within the scope of the appended claims and that are known or customary in the art to which the invention pertains. Therefore, the scope of the claims should not be limited by the preferred embodiments set forth in the description but should be given the broadest interpretation consistent with the overall description.

References

Boase, C. 2001. Bed bugs-back from the brink. Pesticide Outlook 12:159-162.

Jones, S.C. 2004. Bed bugs. Ohio State University Extension FactSheetHYG-2105-04.

Myles, T., B. Brown, B. Bedard, R. Bhooi, K. Bruyere, A.-L. Chua, M. Macsai, R. Menezes, A. Salwan and M. Takahashi. 2003. Bed bugs in Toronto. University of Toronto Center for Urban and Community Studies. Research Bulletin #19.

Potter, M.F. 2012. Bed bugs. University of Kentucky College of Agriculture, Cooperative Extension Service. Entfact-636.

Ter Poorten, M.C. and N.S. Prose. 2005. The return of the common bedbug. Pediatric Dermatology 22:183-187.

Claims (23)

1. A composition comprising an active ingredient, wherein the active ingredient is primarily composed of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone, wherein the composition attracts and/or captures bedbugs when used. 2. The composition according to claim 1, further comprising an effective amount of one or more other compounds. 3. The composition according to claim 2, wherein the additional compound is selected from butyraldehyde, pentanal, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenatenone, ethyl octanoate, methyl octanoate, pentyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L-valine, L-alanine, octanal, nonanal, decanal, (E,E)-2,4-octadienal, (E,Z)-2,4-octadienal, methyl acetate, ( +)-Limonene, (-)-Limonene, 6-Methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, decanoic acid, myristic acid, androstenone, 3-methylindole, 1-eicosadiol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol. 4. The composition according to claim 2 or 3, wherein the mixture of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone with an effective amount of said one or more additional compounds is formulated as a sustained-release feed, wherein said one or more additional compounds are selected from butyraldehyde, pentanal, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenafenone, ethyl octanoate, methyl octanoate, pentanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L-valine, L-alanine, octanal, nonanal, and decanal. (E,E)-2,4-octadienal, (E,Z)-2,4-octadienal, benzyl acetate, (+)-limonene, (-)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, decanoic acid, myristic acid, androstenone, 3-methylindole, 1-eicosadiol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol. 5. The composition according to claim 1, wherein it is formulated as granules, powder, dust, paste, gel, suspension, emulsion or liquid solution. 6. The composition according to claim 1, wherein the mixture of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone is formulated as a sustained-release bait. 7. The composition according to claim 1, wherein it is formulated as a sustained-release bait. 8. A composition comprising an active ingredient, wherein the active ingredient of the composition is primarily composed of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone in combination with an effective amount of one or more additional compounds, said one or more additional compounds being selected from butyraldehyde, pentanal, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenatenone, ethyl octanoate, methyl octanoate, pentyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L-valine, L-alanine, octanal, nonanal, decanal, (E,E)-2, 4-Octadienal, (E,Z)-2,4-Octadienal, benzoyl acetate, (+)-limonene, (-)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, decanoic acid, myristic acid, androstenone, 3-methylindole, 1-eicosadiol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol, wherein the composition, when used, attracts and/or traps bedbugs. 9. A method for attracting and/or capturing bedbugs, comprising providing the composition according to claim 1 or 8 at a desired location. 10. The method of claim 9, wherein dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone are provided in the sustained-release device, the sustained-release device comprising a permeable sealed reservoir loaded with dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone. 11. The method of claim 9, wherein dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone, and one or more other compounds are provided in the sustained-release device, said sustained-release device comprising a permeable sealed reservoir loaded with dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone, and said one or more other compounds, wherein said one or more other compounds are selected from butyraldehyde, pentanal, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenatenone, ethyl octanoate, methyl octanoate, pentanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxyl... Kynurenine O-sulfate, L-valine, L-alanine, octaldehyde, nonanal, decanal, (E,E)-2,4-octadienal, (E,Z)-2,4-octadienal, benzyl acetate, (+)-limonene, (-)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, decanoic acid, myristic acid, androstenone, 3-methylindole, 1-eicosadiol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol. 12. The method of claim 9, wherein the bedbug is the common bedbug Cimex lectularius or the tropical bedbug C. hempterus. 13. The method of claim 9, wherein the desired location is within, above, or near the bed bug control device. 14. The method of claim 13, wherein the bedbug control device is a detector, a monitor, or a trap. 15. The method of claim 14, wherein the slow-release device of any one of claims 10 or 11 is disposed in the detector, monitor or trap. 16. The method of claim 9, further comprising combining the composition with a heat source. 17. The method of claim 9, further comprising combining the composition with a carbon dioxide source. 18. The method of claim 9, further comprising combining the composition with a bedbug lethal insecticide. 19. Applications of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal and 2-hexanone for attracting and/or capturing bed bugs. 20. An application of dimethyl disulfide, dimethyl trisulfide, (E)-2-hexenal, (E)-2-octenal, and 2-hexanone in combination with effective amounts of one or more other compounds for attracting and/or capturing bedbugs, wherein the one or more other compounds are selected from butyraldehyde, pentanal, hexanal, benzaldehyde, benzyl alcohol, acetophenone, verbenatenone, ethyl octanoate, methyl octanoate, pentyl hexanoate, dimethylaminoethanol, N-acetylglucosamine, L-3-hydroxykynurenine O-sulfate, L-valine, L-alanine, octanal, nonanal, decanal, (E,E)- 2,4-Octadienal, (E,Z)-2,4-Octadienal, benzoyl acetate, (+)-limonene, (-)-limonene, 6-methyl-5-hepten-2-one (sulcatone), geranylacetone, carbon dioxide, 1-octen-3-ol, L-carvone, L-lactic acid, propionic acid, butyric acid, valeric acid, oleic acid, palmitic acid, stearic acid, linoleic acid, lauric acid, decanoic acid, myristic acid, androstenone, 3-methylindole, 1-eicosadiol, pentadecanoic acid, squalene, cholesterol, and 2,2-dimethyl-1,3-dioxolane-4-methanol. 21. The application according to claim 19 or claim 20, wherein the bedbug is the common bedbug Cimexlectularius or the tropical bedbug C. hempterus. 22. The application of the composition according to claim 1 for attracting and/or capturing bedbugs. 23. The application of the composition according to claim 1 for attracting and/or capturing common bed bug Cimex lectularius or tropical bed bug C. hempterus.