CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of the filing of U.S. Provisional Patent Application No. 63/256,846, entitled "Insect Repellent and Bio-Pesticide and Method of Testing", filed on October 18, 2021, and the specification and claims thereof are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention (Technical Field):

[0002] Embodiments of the present invention relate to insecticide and insect repellent compositions and methods of and systems for testing the same. In particular, insecticide and insect repellent compositions including guayule resin.

Description of Related Art:

[0003] Natural rubber is harvested from the hevea tree (Hevea brasiliensis). Guayule (Parthenium argentatum) is a promising alternative to hevea rubber. Guayule is a perennial desert shrub native to the southwestern United States and northern Mexico and is one of the main alternative sources of high-quality natural rubber. To improve the economics of the cultivation and processing of guayule as a source of rubber, development of value-added uses for co-products of guayule rubber processing, including guayule resin and bagasse, are desirable. Guayule resin is rich in some potentially valuable secondary metabolites such as sesquiterpenes, triterpenoids, and fatty acids. The secondary metabolites may also include sesquiterpene esters, triterpenoids, sterols, and triacylglycerols. [0004] Synthetic pesticides are used globally for crop protection, but overuse and misuse of synthetic pesticides can resuit in harmfui effects on humans and the environment and may be toxic to non-target organisms. Despite the efficaciousness of synthetic pesticides, continuous use has brought challenges such as the development of pesticide resistant pests. Moreover, some synthetic pesticides may not be easily biodegradable. There has been increased demand for bio-pesticide products for pest management as an alternative to use of synthetic pesticides. One of the possible applications of guayule resin is as a bio-pesticide.

[0005] The present application relates to an insect repellent. The use of repellents is the most efficient method to prevent disease transmission by insects and for controlling insect presence. Common synthetic repellents include DEET (N,N-diethyl-meta-toluamide), IR3535 (ethyl butylacetylaminopropionate), icaridin (picaridin), DEPA (N,N-diethylphenylacetamide), and permethrin (synthetic pyrethroid). Botanical insect repellents are pest management products made primarily from plant materials and extracts, and are considered an alternative to synthetic repellents. The most used botanically derived insect repellents are essential oils such as lemon eucalyptus oil, andiroba, neem oil, and citronella. Botanical insect repellents may suffer from a number of disadvantages, including a rapid volatilization period, short action time, and strong odor.

[0006] Guayule resin compounds have insect repellent activity and the resin can be used as repellent. Guayule resin contains mono- and sesquiterpenes, and their oxygenated derivatives, such as a- and p~ pinene, D- limonene, β-phellandrene, elemene, and β- eudesmol. These chemicals are among the active compounds for insect repellents currently in use. Triterpenoids, such as argentatins, are another major group of compounds in guayule resin that have repellent activity.

[0007] The cockroach can be found globally, and it is of interest due to its widespread distribution and its adaptability to many different habitats. The German cockroach is considered an urban nuisance pest. It is the most widespread and common cockroach in urban areas and is considered one of the most relevant household pests because of its prevalence and proximity to human residences. Cockroach allergens can cause individuals to exhibit allergic reactions, including asthma attacks. The German cockroach can spread and thrive so spectacularly because of its resistance to a majority of insecticides. Conventional cockroach control consists of using commercial insecticides and repellents, such as fipronil and DEET. These insecticides and repellents may be toxic to different species and due to the increasing resistance of German cockroaches to them, there is a need for new natural and organic insecticides and repellents and/or development of other methods to kill or repel them.

[0008] The present invention provides an alternative to existing insecticides and repellants, as well as a method for testing of such insecticides and/or repellants. The compositions of the present invention have efficacy as an insect repellent for urban pest insects such as cockroaches and bed bugs. The use of guayule resin derived from guayule rubber processing as an insect repellent helps the economic viability of guayule as an industrial crop. Guayule is expected to be a low-cost insect repellent because of availability of the source material compared to other insect repellent products.

BRIEF SUMMARY OF EMBODIMENTS OF THE PRESENT INVENTION

[0009] The present invention generally relates to a system for testing an insect repellent, the system comprising: a vessel comprising a bottom surface; a test material receiver in communication with said bottom surface; a test material attached to said test material receiver; and a first harborage at least partially disposed over said test material receiver. In another embodiment, the insect repellant is a cockroach repellent. In another embodiment, the insect repellent comprises guayule resin. In another embodiment, the insect repellent comprises a guayule resin fraction, in another embodiment, the system further comprises a second harborage. In another embodiment, the system further comprises a control at least partially disposed beneath said second harborage.

[0010] The present invention also relates to method for testing an insect repellent, the method comprising: providing a vessel comprising a bottom surface; attaching a test material receiver to the bottom surface; attaching a test material to the test material receiver; and at least partially disposing a first harborage at least partially disposed over the test material receiver. In another embodiment, the insect is a cockroach. In another embodiment, the insect repellent comprises guayule resin. In another embodiment, the insect repellent comprises a guayule resin fraction. In another embodiment, the method further comprises providing a second harborage. In another embodiment, the method further comprises at least partially disposing a control beneath the second harborage.

[0011] The present invention also relates to a composition for repelling an insect, the composition comprising: a guayule resin fraction comprising a terpene, a lipid, and a fatty acid. In another embodiment, the guayule resin fraction is aged. In another embodiment, the terpene comprises a sesquiterpene. In another embodiment, the terpene comprises an oxygenated sesquiterpene. In another embodiment, the lipid comprises a medium chain lipid. In another embodiment, the fatty acid comprises a medium chain fatty acid. In another embodiment, the composition further comprises an added lipid. In another embodiment, the composition further comprises a solvent.

[0012] Objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate one or more embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The drawings are only for the purpose of illustrating one or more embodiments of the invention and are not to be construed as limiting the invention. In the drawings:

[0014] Fig. 1 is a schematic showing the setup of a “tent on the side” bioassay for evaluation of guayule resin fraction repellent activity;

[0015] Fig. 2 is a graph showing the Fourier-transform ion cyclotron resonance (“FT- ICR”) mass spectrometry results for whole guayule resin; [0016] Fig. 3 is a graph showing the chemical components of whole guayule resin;

[0017] Fig. 4 is a flow diagram showing the solvent extraction method for guayule resin;

[0018] Fig. 5 is a diagram showing the test area and parameters for preliminary cockroach repellency tests using guayule resin fractions;

[0019] Fig. 6 is a series of heat maps showing movement of an individual cockroach over a 25 minute period in the arena;

[0020] Fig. 7 is a heat map showing movement of an individual cockroach over a 10 minute period in an untreated area;

[0021] Fig. 8 is a series of heat maps showing validation tests for small fractions for guayule resin fractions prepared by solvent extraction;

[0022] Fig. 9 is a series of heat maps and associated schematics showing validation tests for small fractions for guayule resin fractions prepared by solvent extraction and for whole resin;

[0023] Fig. 10 is a diagram showing a test area with a harborage treated with 10% Java oil and a harborage over the treated test material;

[0024] Fig. 11 is a diagram showing the arrangement of an arena with two harborages and two testing zones;

[0025] Fig. 12 is a table showing repellency test results for DEET, whole guayule resin, and a control against German cockroach; [0026] Fig. 13 is a graph showing the repellent rate of treatments for DEET, whole guayule resin, and a control against German cockroach;

[0027] Fig. 14 is a graph showing the distance to treated zone measurements of lasting effect experiments of cockroaches interacting with areas treated with fresh and aged guayule resin fractions;

[0028] Fig. 15 is a graph showing the number of visits to treated zone results of lasting effect experiments of cockroaches interacting with areas treated with fresh and aged guayule resin fractions;

[0029] Fig. 16 is a graph showing the duration of treated zone results of lasting effect experiments of cockroaches interacting with areas treated with fresh and aged guayule resin fractions;

[0030] Fig. 17 is a graph showing the latency results of lasting effect experiments of cockroaches interacting with areas treated with fresh and aged guayule resin fractions;

[0031] Fig. 18 is a graph showing the distance to treated zone measurements results of behavioral tests of cockroaches interacting with areas treated with guayule resin fractions;

[0032] Fig. 19 is a graph showing the duration of treated zone results of behavioral tests of cockroaches interacting with areas treated with guayule resin fractions;

[0033] Fig. 20 is a graph showing the number of visits to treated zone results of behavioral tests of cockroaches interacting with areas treated with guayule resin fractions;

[0034] Fig. 21 is a graph showing the velocity results of behavioral tests of cockroaches interacting with areas treated with guayule resin fractions;

[0035] Fig. 22 is a graph showing the latency results of behavioral tests of cockroaches interacting with areas treated with guayule resin fractions;

[0036] Fig. 23 is a heat map showing the composition of guayule resin fractions; [0037] Fig. 24 is a graph showing the results of behavioral tests of cockroaches interacting with areas treated with guayule resin fractions;

[0038] Fig. 25 is graph of multidimensional clustering analysis with similarity cutoff 0.7;

[0039] Fig. 26 is a graph showing the repellency activity results of behavioral tests of cockroaches interacting with areas treated with guayule resin fractions;

[0040] Fig. 27 is a heat map showing the results of structural similarity between guayule resin compounds and known active insecticide and/or repellent compounds; and

[0041] Fig. 28 is a heat map showing the results of physicochemical property similarity between 22 guayule resin compounds and 27 known active insecticide and/or repellent compounds.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention relates to a method for testing the insect repellent activity of a guayule resin fraction, the method comprising: providing a vessel comprising a bottom surface; attaching a material to the bottom surface; applying a test material comprising a guayule fraction to a test material receiver; at least partially disposing a first harborage over the test material receiver; at least partially disposing an insect into the vessel; and observing the movement of the insect. The test solution may be applied to a first end of the bottom surface. The insect may be disposed into the vessel at a second end of the bottom surface. The method may further comprise providing a control and a second harborage. The second harborage may be at least partially disposed over the second harborage. The test material may be aged.

[0043] The present invention relates to a composition comprising guayule resin fraction. The guayule resin may be a primarily non-rubber extract of the guayule plant. The guayule resin may be fractionated to form guayule resin fractions. The guayule resin may be fractionated by solvent extraction, vacuum distillation, or a combination thereof. The guayule resin may comprise a mixture of compounds. The guayule resin compounds may be individually applied or be applied as a part of a guayule resin fraction after guayule resin fractionation.

[0044] The present invention also relates to a system for testing the insect repellent activity of a guayule resin fraction, the system comprising: a vessel comprising a bottom surface; a material attached to the bottom surface of the vessel; a test material comprising a guayule resin fraction; a test material receiver; and a first harborage. The bottom surface may comprise a first end and a second end and the test material may be aged. The system may also comprise a control. The system may further comprise a second harborage. The second harborage may be at least partially disposed over the control. The first and/or second harborage may comprise an absorbent material. The absorbent material may comprise paper.

[0045] The terms “resin” or “whole resin” shall mean whole guayule resin unless otherwise indicated by the description.

[0046] The term “insect” shall mean any insect, arachnid, or mobile invertebrate. The terms “vessel” and “arena” are synonymous throughout the description.

[0047] The term “zone” means an area of the vessel or arena. An area, region, or zone of the vessel or arena may be untreated or treated.

[0048] The composition, system, and method of the present invention may be used to prepare insect repellent compounds comprising guayule resin. The advantages of the present invention include, but are not limited to, improving the economic efficiency of rubber extraction from guayule by providing a beneficial coproduct; providing a low-cost, natural insect repellent; and providing an insect repellent that lasts longer than a synthetic, commercially available insect repellent. The insect repellent composition of the present invention may last longer than other insect repellents by at least two days and have greater repellent activity over time compared to other insect repellents. The added benefit of manufacturing and selling the insect repellent composition of the present invention may allow rubber harvesting from guayule to be the same or lower in cost compared to traditional rubber manufacturing. [0049] Turning now to the drawings, Fig. 1 shows the setup of a “tent on the side” bioassay for evaluation of guayule resin fraction repellent activity. Testing system 10 comprises vessel 12 having diameter 14. Harborage 22 is disposed within vessel 12 and comprises length 18 and width 20. Harborage 22 may be prepared from material 26 having side edges 28, 30, and end 24, which may allow material 26 to be folded. Insect 16 may be disposed within testing system 10.

[0050] Fig. 2 shows the Fourier-transform ion cyclotron resonance (“FT-ICR”) mass spectrometry results for whole guayule resin. O ₂ , O ₃ , and O ₄ heteroatoms are most abundant.

[0051] Fig. 3 shows chemical components of whole guayule resin. Monoterpenes, oxygenated terpenes, and sesquiterpene esters are present in the resin.

[0052] Fig. 4 shows the solvent extraction method for guayule resin. Solvent extraction yields terpene-rich, lipid, and low-molecular weight fractions.

[0053] Fig. 5 shows the test area and parameters for preliminary cockroach repellency tests using guayule resin fractions. Distance from treated area, latency, movement velocity, number of visits to treated area, and duration in treated area are measurable.

[0054] Fig. 6 shows movement of an individual cockroach over a 25 minute period in the arena. The individual cockroach did not move near fractions E, F, J, or near Java during the period.

[0055] Fig. 7 shows movement of an individual cockroach over a 10 minute period in an untreated area and treated area. The individual cockroach moved freely in untreated areas but did not enter into areas treated with resin or Java during the period.

[0056] Fig. 8 shows validation tests for small fractions for guayule resin fractions prepared by solvent extraction. Cockroaches do not enter region near whole resin or a terpene-rich resin fraction. [0057] Fig. 9 shows validation tests for small fraction for guayule resin fractions prepared by solvent extraction and for whole resin. The lipid fraction was least effective in controlling the movement of the cockroach.

[00S8] Fig. 10 shows a test area with a harborage treated with 10% Java oil and a harborage over the treated test material. Testing system 32 may be split along midline 38 into treated region 34 and untreated region 36. Insect behavior may be evaluated by assessing insect movement and movement speed to and from treated region 34 and untreated region 36 as well as time spent in treated region 34 and untreated region 36.

[0059] Fig. 11 shows the arrangement of an arena with two harborages and two testing zones. Testing system 40 comprises harborages 44 and 46 disposed within vessel 42. Harborage 44 may be disposed above a treated material and harborage 46 may be disposed above a control.

[0060] Fig. 12 shows repellency test results for DEET, whole guayule resin, and a control. Resin has comparable repellency characteristics to DEET.

[0061] Fig. 13 shows the repellent rate of treatments for DEET, whole guayule resin, and a control.

[0062] Fig. 14 shows the distance to treated zone measurements of lasting effect experiments of cockroaches interacting with areas treated with fresh and aged guayule resin fractions. Aged fraction J has a similar distance to treated zone measurement compared to fresh fraction J.

[0063] Fig. 15 shows the number of visits to treated zone results of lasting effect experiments of cockroaches interacting with areas treated with fresh and aged guayule resin fractions. Aged fractions E and J have greater numbers of visits to the treated zone compared to fresh fractions E and J, but less than aged Java.

[0064] Fig. 16 shows the duration of treated zone results of lasting effect experiments of cockroaches interacting with areas treated with fresh and aged guayule resin fractions. Aged fraction J has a similar duration in treated zone measurement compared to fresh fraction J.

[0065] Fig. 17 shows the latency results of lasting effect experiments of cockroaches interacting with areas treated with fresh and aged guayule resin fractions. Aged fraction J has a similar latency measurement compared to aged Java.

[0066] Fig. 18 shows the distance to treated zone results of behavioral tests of cockroaches interacting with areas treated with nine guayule resin fractions. Fractions E, F, and J have similar distance to treated zone measurements compared to Java.

[0067] Fig. 19 shows the duration of treated zone results of behavioral tests of cockroaches interacting with areas treated with nine guayule resin fractions. Fractions E, F, and H have similar duration in treated zone measurements compared to Java.

[0068] Fig. 20 shows the number of visits to treated zone results of behavioral tests of cockroaches interacting with areas treated with nine guayule resin fractions. Fractions E, F, H, and J, have similar number of visits to treated zone measurements compared to Java.

[0069] Fig. 21 shows the velocity results of behavioral tests of cockroaches interacting with areas treated with nine guayule resin fractions. Fractions B, E, H, and J have similar velocity measurements compared to Java.

[0070] Fig. 22 shows the latency results of behavioral tests of cockroaches interacting with areas treated with nine guayule resin fractions. Fractions B, E, H, and J have similar latency measurements compared to Java.

[0071] Fig. 23 shows the composition of guayule resin fractions. Fraction E has high levels of sesquiterpenes compared to most of the other fractions. Fraction J has high levels, of oxygenated sesquiterpenes and medium chain fatty acids compared to most of the other fractions. [0072] Fig. 24 shows the results of behavioral tests of cockroaches interacting with areas treated with guayule resin fractions. The terpene-rich fraction performed most similarly to Java.

[0073] Fig. 25 shows a scatter plot of multidimensional clustering analysis with similarity cutoff 0.7. The scatter plot shows the most abundant compounds in the guayule resin. Group 1, 2, 3, 4, and 6 have properties similar to known insecticides and/or repellents.

[0074] Fig. 26 shows the results of behavioral tests of cockroaches interacting with areas treated with guayule resin fractions. Fractions E and J show similar repellency activity to Java.

[0075] Fig. 27 shows the results of structural similarity among guayule resin compounds and known active insecticide and/or repellent compounds. 22 guayule resin compounds and 27 known active insecticide and/or repellent ingredients were compared using the hierarchical clustering method. The guayule resin compounds contain similar or identical ingredients to known active insecticide and/or repellent ingredients.

[0076] Fig. 28 shows the results of physicochemical property similarity among 22 guayule resin compound and 27 known active insecticide and/or repellent compounds. 22 guayule resin compounds and 27 known active insecticide and/or repellent ingredients were compared using hierarchical clustering method.. The guayule resin compounds contain structural similarity to known active insecticide and/or repellent ingredients.

[0077] The guayule resin fraction may be an insect repellent. The guayule resin fraction may repel insects including, but not limited to, cockroaches, bed bugs, kissing bugs, mosquitos, arachnids, or other insects or pests. The cockroaches may be Turkestan cockroaches (Shelfordella lateralis), German cockroaches (Blatella germanica), or others. The bed bugs may be Cimex lectularius. The kissing bugs may be triatominae or others. The arachnids may be scorpions, spider, or others. [0078] The guayule resin compounds may be individually applied or be applied as a part of a guayule resin fraction after guayule resin fractionation. Guayule resin fractions may comprise solvent-extracted fractions and/or vacuum distillate fractions. Solvent-extracted fractions may be obtained by dissolving the resin in ethanol, methanol, or another organic solvent. The solvent-extracted fraction may be dissolved at a temperature of at least about 30 °C, about 30 °C to about 35 °C, about 35 °C to about 40 °C, about 40 °C to about 45 °C, about 45 °C to about 50 °C, about 50 °C to about 55 °C, about 55 °C to about 60 °C, or about 60 °, and may be dissolved by stirring the resin to complete the dissolution of the resin. The solvent-extracted fractions may be cooled to at least about -10 °C, about -10 °C to about -5 °C, about -5 °C to about 0 °C, about 0 °C to about 5 °C, about 5 °C to about 10 °C, or about 10 °C. The solvent-extracted fractions may comprise waxes, fats, low molecular weight rubber (“LMWR”), polar components, e.g., defatted resin, or a combination thereof. Fats and defatted resin may be separated in a separatory funnel. Solvent, i.e., ethanol, in defatted resin may be evaporated. The evaporation may be performed by a vacuum rotary evaporator. The defatted resin may comprise terpenes. The ratio of resin (dry weight g) to solvent (volume in liters) may be at least about 50 g/l, about 50 g/l to about 75 g/l, about 75 g/l to about 100 g/l, about 100 g/l to about 125 g/l, about 125 g/l to about 150 g/l, about 150 g/l to about 175 g/l, about 175 g/l to about 200 g/l, or about 200 g/l.

[0079] The insects may be obtained from a colony. The colony may be maintained at about 20 °C, about 20 °C to about 25 °C, about 25 °C to about 30 °C, about 30 °C to about 35 °C, or about 35 °C. The colony may be maintained at a relative humidity at about 0%, about 0% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, or about 100%. The colony may be maintained for a photoperiod of about 8 h:16 h (light: dark), about 10 h:14 h (light: dark), about 12 h:12 h (light: dark), about 14 h:10 h (light: dark), or about 16 h:8 h (light: dark). The colony may be kept for four days) in an incubator for acclimation. [0080] Guayule resin fractions may be obtained from guayule resin by a vacuum distillation method. Guayule resin fractions may be diluted to at least about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, or about 25% by weight in acetone, or any other organic solvent.

[0081] The method for testing the insect repellent activity of a guayule resin fraction may comprise a control. The control may be a positive control. The positive control may comprise Java. The positive control may comprise a concentration of at least about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, or about 25% by weight of Java in a solvent. The solvent may comprise acetone. The control may be a negative control. The negative control may comprise acetone.

[0082] The vessel may contain insects. The vessel may comprise a Petrie dish, flask, bowl, beaker, box, or any other container with a bottom and a side to form a cavity. The vessel may comprise a treated zone, an untreated zone, or a combination thereof. The system may comprise a vessel for controls and to test guayule fractions for insect repellent activity. The position of each vessel and the direction of the treated zone may be randomized.

[0083] The test material receiver may comprise an absorbent material. The material may comprise paper. The paper may comprise filter paper. The test material receiver may receive test material. The test material receiver may comprise an area of the arena and may comprise an area of any size, but preferably at least about 8 mm x 12 mm, at least about 1 cm ² , or at least about 2 cm ² . The test material receiver may be the edge of or be a portion of a larger material. The test material may comprise a solution, mixture, or a combination thereof. The test material may comprise a guayule resin fraction. The test material may comprise a solution of at least about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, or about 25% by weight of guayule resin fraction. The test material may be applied evenly to the test material receiver. The volume of the test material may be at least about 5 μL, about 5 μL to about 10 μL, about 10 μL to about 15 μL, about 15 μL to about 20 μL, about 20 μL to about 25 μL, about 25 μL to about 30 μL, about 30 μL to about 35 μL, about 35 μL to about 40 μL, about 40 μL to about 45 μL, about 45 μL to about 50 μL, or about 50 μL. The test material may be applied to the test material receiver at a concentration of at least about 0.1 mg/cm ² , about 0.1 mg/cm ² to about 0.5 mg/cm ² , about 0.5 mg/cm ² to about 1.0 mg/cm ² , about 1.0 mg/cm ² to about 1.5 mg/cm ² , about 1.5 mg/cm ² to about 2.0 mg/cm ² , about 2.0 mg/cm ² to about 3.0 mg/cm ² , about 3.0 mg/cm ² to about 4.0 mg/cm ² , about 4.0 mg/cm ² to about 5.0 mg/cm ² , about 5.0 mg/cm ² to about 6.0 mg/cm ² , about 6.0 mg/cm ² to about 7.0 mg/cm ² , about 7.0 mg/cm ² to about 8.0 mg/cm ² , about 8.0 mg/cm ² to about 9.0 mg/cm ² , about 9.0 mg/cm ² to about 10.0 mg/cm ² , or about 10.0 mg/cm ² . The test material may be dried for at least about 15 min. to about 20 min. The system may also comprise a control region. The control region may comprise an absorbent material. The control region may receive a control. The control may be a positive control or negative control. The system may further comprise material attached to the bottom of the vessel.

[0084] The harborage may be folded and/or manipulated into a shape. The harborage may be black, white, or any other color. The harborage may comprise a tent. The harborage may comprise dimensions of at least about 8 mm x 12.5 mm x 6 mm. The harborage may be at least partially disposed over the test material receiver or an untreated and/or control region. The system may comprise a first harborage at least partially disposed over the test material receiver and a second harborage at least partially disposed over an untreated and/or control region. The harborage and control region may be two attracting forces that help compare the strength of the repelling force of test materials.

[0085] The system for testing the insect repellent activity of a guayule resin fraction may comprise a camera. The camera may be near infra-red (“NIR”), infra-red (“IR”), high- resolution, monochrome, or a combination thereof. The system for testing the insect repellent activity of a guayule resin fraction may also comprise data collection software. The data collection software may capture video images and/or track the activity of insects in the arena. The data collection software may generate heat maps showing movement of an individual roach over a time period in the arena. The time period may be at least about 10 min., about 10 min. to about 15 min., about 15 min. to about 20 min., about 20 min. to about 25 min., about 25 min. to about 30 min., or about 30 min.

[0086] The system for testing the insect repelient activity of a guayule resin fraction may comprise a lining to increase the height of the vessel. The lining may comprise paper or other material. The paper may comprise acetate paper. The lining may prevent insects from escaping the arena and/or prevent glare in the recorded video. The system for testing the insect repellent activity of a guayule resin fraction may also comprise an insect barrier. The insect barrier may be applied to the inner walls of the vessel and/or lining. The insect barrier may prevent insects from escaping the arena.

[0087] The guayule resin fractions may act as pesticides. The guayule resin fractions may affect the locomotor activity of insects. The guayule resin fractions may have a long action time compared to other pesticides and/or insect repellents. The guayule resin fractions may not evaporate or may evaporate more slowly than other pesticides and/or insect repellents. The guayule resin fractions may be modified by polymeric microencapsulation.

[0088] The method for testing the insect repellent activity of a guayule resin fraction may comprise release of an insect into the arena. The insect may be released onto the opposite edge of the test material receiver. The method may further comprise at least partially disposing an insect into a shell vial. The shell vial may be at least about 5 mm, about 5 mm to about 10 mm, about 10 mm to about 15 mm, about 15 mm to about 20 mm, or about 21 mm in diameter. The shell vial may be at least about 10 mm, about 10 mm to about 20 mm, about 20 to about 30 mm, about 30 mm to about 40 mm, about 40 mm to about 50 mm, about 50 mm to about 60 mm, about 60 mm to about 70 mm, about 70 mm to about 80 mm, about 80 mm to about 90 mm, about 90 mm to about 100 mm, or about 100 mm in height. The method may also comprise inverting the shell vial into the arena. The method may also comprise lifting the shell vial off of the insect. [0089] The method for testing the insect repellent activity of a guayule resin fraction may comprise acclimating the insect in the arena for at least about 5 min., about 5 min. to about 10 min., about 10 min. to about 15 min, about 15 min. to about 20 min., about 20 min. to about 25 min., about 25 min. to about 30 min., or about 30 min. The method may be performed for a duration of at least about 5 min., about 5 min. to about 10 min., about 10 min. to about 15 min., about 15 min. to about 20 min., about 20 min. to about 25 min, about 25 min to about 30 min., or about 30 min.

[0090] The method for testing the insect repellent activity of a guayule resin fraction may comprise measuring variables from arenas comprising guayule resin fractions and/or a control arenas. The variables include, but are not limited to, elapsed time until first visit to the treated zone; number of visits to the treated zone; percent of time spent in the treated zone; distance traveled in the treated zone; velocity in the treated zone; or a combination thereof.

[0091] The method for testing the insect repellent activity of a guayule resin fraction may comprise recording insect activity. Insect activity may be recorded with a camera, data collecting software, or a combination thereof.

[0092] The guayule resin fraction may be analyzed by FT-ICR mass spectrometry. FT- ICR mass spectrometry may be used to measure the mass to charge ratio because the angular velocity of the ions is dependent on their mass. The advantages of FT-ICR mass spectrometry are increased resolution, sensitivity, mass accuracy and fast duty cycle. Frequencies may be measured very accurately. In FT-ICR mass spectrometry those frequencies may be repeated measured with multiple excitation pulses. FT-ICR mass spectrometers achieve resolutions in the 10 ⁶ range. Currents induced by the cycling ions may be amplified. FT-ICR mass spectrometry cells may detect as little as one ion and can store up to about 10 ⁵ ions. Mass accuracy may be about 0.01 Da. Mass spectra may be collected in milliseconds to seconds. FT-ICR mass spectrometry may allow assignment of a unique elemental composition to each ion signal observed in complex mass spectra. FT-ICR mass spectrometry may analyze complex mixtures, e.g., large macromolecules, with very close mass to charge ratios to each other.

[0093] The composition comprising a guayule resin fraction may comprise compounds including, but not limited to, a terpene, a lipid, and a fatty acid. The terpene may include, but is not limited to, a monoterpene, an oxygenated monoterpene, a sesquiterpene, an oxygenated sesquiterpene, or a combination thereof. The lipid may include, but is not limited to, a short lipid, a medium chain lipid, a long chain lipid, or a combination thereof. The short chain lipid may comprise a carbon chain between about 13 to about 17 carbons in length. The medium chain lipid may comprise a carbon chain between about 18 and 20 carbons in length. The long chain lipid may comprise a carbon chain of about 20 carbons or more, or more than 20 carbons. The fatty acid may include, but is not limited to, a short chain fatty acid, a medium chain fatty acid, a long chain fatty acid, or a combination thereof. The short chain fatty acid may comprise a carbon chain between about 13 to about 17 carbons in length. The medium chain fatty acid may comprise a carbon chain between about 18 and 20 carbons in length.

The long chain fatty acid may comprise a carbon chain of at least about 20 carbons.

[0094] The guayule resin fraction may comprise at least about 30%, about 30% to about 80%, about 40% to about 70%, about 50% to about 60%, about 80% sesquiterpenes by weight. The guayule resin fraction may also comprise at least about 40%, about 40% to about 80%, about 50% to about 70%, about 55% to about 65%, or about 80% oxygenated sesquiterpenes by weight. The guayule resin fraction may further comprise about 0.3%, about 0.3% to about 2.5%, about 0.5% to about 2.0%, about 1.0% to about 1.5%, or about 2.0% medium chain fatty acids by weight.

[0095] The guayule resin fraction may comprise compounds including, but not limited to, santolina triene, thujene<alpha->, pinene<alpha->, camphene, thuja-2, 4(10)-diene, benzaldehyde, sabinene, pinene<beta->, hepten-2-one<6-methyl-5->, myrcene, carene<delta- 2->, mentha-1(7),8-diene<para->, cymene<ortho->,sylvestrene, phellandrene<beta->, ocimene<(E)-beta->, terpinene<gamma->, terpinolene, dimethyl styrene<2,5->, linalool, mentha-2,8-dien-1-o!<trans-para->, campholenal<alpha->, ocimene<neo-allo->, sabinol<trans- > (trans for OH vs. IPP), isocitral<Z->, pinocamphone<trans->, mentha-1,5-dien-8-ol<para->, borneol, terpinen-4-ol, cymen-8-ol<para->, acetophenone <para-methyl->, cryptone, myrtenal, isophorone<4-methlyene->, carveol<cis->, carvacrol (methyl ether), cumin aldehyde, carvone, necrodol acetate<trans-alpha->, bornyl acetate, cymen-7-ol<para->, pinocarvyl acetate<cis->, cycloisolongifolene<didehydro->, presilphiperfol-7-ene, menth-1-en-7-a!<3-oxo-para->, elemene<delta->, patchoulene<beta->, cubebene<alpha->, longicyclene, copaene<alpha->, cubebene<beta->, modheph-2-ene, isocomene<alpha->, funebrene<alpha->, duprezianene<beta->, aromadendrene, guaiadiene<6,9->, amorpha-4,11-diene, aromadendrene<allo->, dauca-5,8-diene, humulene<alpha->, acoradiene<beta->, thujopsadiene, gurjunene<gamma->, amorphene<gamma->, selinene<beta->, curcumene<ar- >, muurola-4(14),5-diene<trans->, bulnesene<alpha->, cadinene<gamma->, nootkatene, zonarene, calamenene<cis->, calacorene<beta->, germacrene B, elemol, nerolidol<E->, viridiflorol, copaen-4-alpha-ol<beta->, khusimone, eudesmol<alpha->, valerianol, bulnesol, santalol<(Z)-alpha->, cadalene, oplopanone, carissone, or a combination thereof.

[0096] The guayule resin fractions may comprise secondary metabolites. The secondary metabolites may have insect repellent activity. The secondary metabolites may comprise sesquiterpenes, triterpenoids, fatty acids, sesquiterpene esters, triterpenoids, sterols, triacylglycerols, or a combination thereof.

Industrial Applicability:

[0097] The invention is further illustrated by the following non-limiting examples.

Example 1

[0098] Behavioral tests were conducted in thirteen 90 mm Petri dishes. Eleven dishes were each treated with a single guayule resin fraction, one dish with a positive control, and one dish with a negative control. The position of each arena and the direction of treated zone, e.g., north, south, west, and east, were randomized in each trial. [0099] For each arena, a piece of filter paper was attached to the bottom of each Petrie dish. Then a rectangular area (8 mm* 12 mm) dose to the edge of the filter paper was impregnated evenly with 10 μL of the 10% by weight test material and left to dry under a fume hood for around 15-20 min. A filter paper harborage with the dimensions of 8 mm by 12.5 mm by 6 mm was placed on the treated area. The design of this bioassay was motivated by the harborage and edges being two attracting forces that help to compare the strength of repelling force of test materials.

[00100] Individual roaches were acclimated to the environment by restricting them in a piece of plastic tubing which was placed in the untreated area for 5 min. Insects were released by lifting up the tubing in each arena. In total, 12 replicates were used along with a common control group for each. Bioassays were conducted under ambient temperature (25 ± 2 °C) and relative humidity (40 ± 10%) during the first three hours into the scotophase, a time in which cockroaches display enhanced locomotor activity.

[00101] A near infrared (“NIR”) camera outfitted with a lens and infrared (“IR”) filter was be used to record cockroach activity in the arenas under dark conditions. The camera was positioned 60-100 cm directly above the center arena. Light for the recordings was provided by two IR illuminators. Software captured video images to track the insect during the 20-25 minute bioassays. The software virtually facilitated the division of each arena into two zones known as “treated” and “untreated.” Multiple variables were be calculated from resin fraction- treated arenas, including elapsed time until first visit to the treated zone; number of visits to the treated zone; percent of time spent in the treated zone; and distance traveled and velocity in treated zone. The same variables were calculated from the activity of cockroaches recorded in control arenas.

[00102] Elapsed time until the first visit to the treated areas and the percent of time spent in the treated areas were analyzed with the nonparametric Mann- Whitney test using Minitab. The number of visits to the treated zones, the distance, and the velocity were analyzed using one-way analysis of variance (ANOVA). The many-to-one comparison was done with Dunnett’s test for mean separation. [00103] Results of three fractions from resin solvent extraction were obtained. Cockroaches significantly avoided the areas treated with Java, whole resin, and terpene-rich fractions, while the avoidance efficacy of lipid fraction was not promising. The cockroaches spent 1.9% and 1.7% of the total time in the treated areas with whole resin and terpene-rich fractions respectively. When compared with that of control arenas, the elapsed time until the cockroaches’ first visit was significantly longer when the area was treated with the whole resin (mean time=118 ±75 s vs. 74.7 ±23.6 s). The visitation rate of cockroaches to the treated area is another parameter indicating the repellency effect of a test material. The results showed the area treated with terpene-rich and whole resin have the lower visitation rate, while the number of visits to the lipid-treated area was not significantly different compared to the negative control ■ mean-15.3 ±1.9 vs. 15.9 ±2.6, respectively; p> 0.05). The cockroaches spent significantly less time in areas treated with terpene-rich and whole resin (mean= 22.7 ± 5.2 s and 28.5 ± 10.7 s, but no significant differences were detected for lipid fraction compared with negative control (mean =47.3 + 8.5 s vs. 60.0 ± 8.0 s, respectively).

[00104] There were also significant differences in velocity between cockroaches in areas treated with resin and terpene-rich fractions (mean= 0.54 ± 0.07 cm/s and 0.51 ± 0.08 cm/s) and those in control areas (mean= 0.41 ± 0.03 cm/s). This parameter is used to understand the effect of test materials on the locomotor activity of cockroaches.

Example 2

[00105] 7.18 g of resin was dissolved in 120 mL of acetone. Less acetone was initially desired to dissolve the resin, but the resin was resistant to dilution in acetone. Due to the resin’s very dense structure, it was very difficult to transfer it from the storage container to a beaker. Sediment still remained upon dilution, and the sediment was removed by decanting off the sediment from the solution. The solution was transported to an urban entomology lab in three 20 ml containers after the 6% by weight solution of resin was separated.

[00106] Bioassays were set up by cutting circular filter paper in half, which would allow each half to receive a different treatment. Instead of using the harborages, tents were used. The test tents were made from black filter paper cut into a rectangle and folded in half. Tents were made from white filter paper for the controls.

[00107] Eighteen different bioassays were used for the test experiment. Six bioassays contained half the filter paper sprayed with DEET and the other half with acetone, six contained half resin and half acetone, and the last six were used as the control with half acetone and half with no treatment. For each bioassay, two tents were used. One was used as a test and one tent was used as a control. The substance that the tent was coated with corresponded to the same compound that was on that side of the filter paper.

[00108] Five cockroaches were released into each bioassay. To gather the cockroaches, 90 cockroaches were removed from the colony and were initially placed into a smaller plastic tub. From there the cockroaches were placed into 18 cylindrical containers, with five cockroaches placed into each container. Before the cockroaches were transferred, the top two cm of each container was lined with polytetrafluoroethylene.

Polytetrafluoroethylene is a slippery substance and prevents the cockroaches from escaping and stops them from attaching to walls.

[00109] Spray bottles were used to add each substance to the filter paper. Six sprays of the compound were added to each filter paper being tested. The spray bottle was weighed before and after spraying to determine the weight of the compound sprayed. The filter papers containing acetone, resin, and DEET were left in a fume hood for approximately an hour to dry. Once they had dried, the filter papers were placed into cylindrical bowls using doublesided paper. The filter paper had to be secured to prevent the cockroaches from crawling underneath the paper and invalidating the experiment. Cross-contamination when handling the paper, including the plain filter paper, was prevented by wearing gloves throughout the process. The data would have otherwise been affected by contaminating the filter paper with human essence because cockroaches are sensitive to human essence.

[00110] The tents were also treated with either DEET, resin, or acetone. 300 μL was added to each tent using a 100 μL pipette. 100 μL was added to the base of the tent, 100 μL was added to the outside of the tent, and 100 μL was added to the inside of the tent. The tents were also stuck to the filter paper in the bioassay to prevent the cockroaches from moving the tents during the course of the experiment.

[00111] The insects were introduced to the bioassay by placing the cylindrical container at a slope so that the cockroaches could crawl out over time. The cockroaches could not be easily placed into the bioassays as they stuck to the surface of the container and could not be dislodged. The cockroaches were then left overnight to allow them time to settle. The cockroaches were checked after 12 hours to see where they were residing.

[00112] The cockroaches were observed at 8:30 AM the next morning and a great number of the cockroaches had escaped due to climbing up the container. The bioassay and the position of the cockroaches was studied and the number of cockroaches under each tent was accounted for. Cockroaches that were out in the open were accounted for. The percentage of cockroaches in the treated tent, in the untreated tent, and outside the tents was calculated for each type of compound, resin, DEET and control. Cockroaches that escaped were not accounted for in the calculations.

Example 3

[00113] The toxicity and avoidance efficacy of non-rubber extracts (resins) for guayule as repellents for urban pest insects was tested. The German cockroach (Blatella germanica), a well-understood model, was used for testing, with the expectation of repeating the experiment with bed bugs (Cimex lectulanus), which have shown a recent resurgence in North America.

[00114] A guayule resin sample was provided from a pilot-scale simultaneous extraction method. A 6% by weight solution of resin was prepared by dissolving 7.18 g into 120 mL of acetone for approximately an hour in a 200 mL beaker. After an hour, some sediment still remained and was removed by decanting the solution. The solution was then separated into three 20 mL containers and sealed to prevent solvent evaporation. [00115] The insects tested were German cockroaches Blatella germanica. The insects were reared in a plastic clear box without exposure to any other insecticide. Three to four days prior to the experiment, approximately 200 cockroaches were selected from a larger colony and placed into a separate container. The container was placed into a light-controlled chamber and the light was set to turn on at 5:00 AM and off and 5:00 PM, allowing for the insects to adjust to the light/dark levels.

[00116] The cockroaches were provided with shelter consisting of cardboard. They were given water from a glass flask fitted with paper towels and were fed animal feed. Water was given in a plastic bottle through a wet paper towel.

[00117] Repellency activity of the guayule resin was investigated using a bioassay. To evaluate the comparative repellency of resin, other compounds were also tested in separate arenas. 2.5% DEET was tested as a positive control. DEET is the most widely used insect repellent, so it was assumed that the cockroaches would avoid areas treated with DEET. Acetone was also tested to act as a negative control. Acetone was present in the resin samples and it was included as a negative control to observe its effect on the cockroaches. The test arenas for the investigation were bowls 12.4 cm diameter by 6.0 cm height containing a sheet of 15 cm Whatman filter paper and two small pieces of filter paper cut and folded into tents. The filter papers were cut into two halves and one half was treated with the testing material, which was DEET, resin, or acetone. The other half was treated with the appropriate control. For the DEET and resin treatment, the control was acetone and for the acetone treatment, the control was no treatment. The experiment required 18 different arenas: six contained half DEET and half acetone, six contained half resin and half acetone, and six contained half acetone and half no treatment. For each arena, the filter paper was treated by using a 118 ml fine mist spray bottle at a distance of approximately 10 cm, resulting in a uniform wet surface at rates of 5.8 mg/cm ² which was equivalent to six sprays. Each arena contained two tents, one treated and one untreated, similar to the filter paper. The tents were treated using a 100 μL pipette and 200 μL of solution was added to each tent. 100μL was added to the base of the tent and 100 μL was added to the outside of the tent. Both the tents and the filter paper were left to dry in a fume hood for an hour. Upon drying, the filter paper was stuck onto the bottom of the bowl using double-sided tape to prevent the cockroaches from burrowing under the filter paper. The tents were stuck onto their appropriate side using a small section of tape, with the treated tent placed on the treated side. Gloves were worn at all times when handling the filter paper to prevent any contamination with human scent to which cockroaches are sensitive. Additionally, petroleum jelly was applied to the top two inches of the glass bowls to prevent cockroach escape.

[00118] In total, 90 German cockroaches were required, with five cockroaches being released into each arena. The cockroaches were removed from the colony using entomological tweezers and placed into smaller cylindrical containers, with five cockroaches being placed in each container. The top two cm of the containers were lined with polytetrafluoroethylene, a slippery substance that prevented the cockroaches from attaching to the wall and escaping. The cockroaches were placed into the arena by placing the cylinders into the arena and leaving them tilted so the cockroaches could crawl from the container into the arena.

[00119] The arenas were left in the dark overnight to allow time for the cockroaches to settle. Twelve hours after treatment, the position of the cockroaches was recorded. The repellent rate was calculated according to the following formula:

[00120] In calculating the repellent rate, the cockroaches that died or escaped were discarded from the results and only those left alive in the arena were used in the calculation. Due to the setup of the arena, some of the cockroaches escaped via the cylindrical tube which was used to transport the cockroaches. However, the percentage repelled could still be calculated by disregarding the number of escaped cockroaches and using the repellent rate.

[00121] The resin had very little repellence effect, having a repellent rate of 6.3%. Almost ail the cockroaches that did not escape were found in the treated half of the arena. There are many potential reasons for this. Firstly, the treated tent was made from black filter paper and this may have been preferable to the cockroaches due to their photophobic nature. Additionally, the percentage of resin in the testing solution was low, being only 6%, and the percentage of essential oils and terpenes was very low, most probably less than 0.3%. The average percentage of essential oils is approximately 1%, therefore, the solution may have shown little efficiency due to the low concentration of certain compounds. Finally, the effect of the treatments was only recorded after 12 hours; the resin may have had some repellence effect which did not extend over the entire time period.

Example 4

[00122] For examples 5 through 10, a Turkestan cockroach colony was established from samples collected at a New Mexico State University (NMSU) feed mill facility in Las Cruces, NM, in 2014. In the laboratory, colonies were maintained in an incubator at 24-25°C, relative humidity of 30-50%, and a photoperiod of 12:12 (L:D) h. Colonies were reared in plastic containers with egg crates used as shelters. Cockroaches were fed on a mixed diet containing rabbit, dog, and cat feed as well as sweetened corn puff cereals (1 : 1 : 1 : 1). Water was given regularly. Fourth-fifth instar nymphs of cockroaches were used because they were the most abundant life stage of the colony.

Example 5

[00123] Experimental arenas and repellency assays with fresh residues were prepared for guayule resin repellent testing. The initial experiment comprised assessing the responses of individual cockroaches to fresh residues of the whole resin, terpene-rich, and lipid-rich in glass Petri plates. Twenty arenas were arranged in blocks in such a way that four were treated with each of the three fractions along with positive controls (four arenas) and negative controls (four arenas). The experiment was repeated three times to complete 20 replicates for each treatment. The position of each arena and the cardinal direction of the treated zone (north, south, west, and east) were randomized in each trial. For each arena, a piece of filter paper was attached to the bottom of each Petri dish. Then, two rectangular filter papers (8 mm*12 mm) were placed close to the edge of the Petri dish, and one of them was treated with the test material, while the other was treated with acetone (control). The filter paper was impregnated evenly with 10 μL of the 10 wt.% of each test material and left to dry under the fume hood for around 15-20 min. Subsequent experiments evaluated the responses of cockroaches to nine vacuum distilled fractions (Fraction including the whole resin (Fraction “A”) and bottom distillation residue (Fraction “L”) fraction, using the same experimental arenas described above. The arenas included 13 Petri dishes, 11 of which were used to evaluate responses to each test fraction while the other two were used for positive controls (java citronella oil) and negative controls (acetone). The experiment was repeated 12 times. Individual cockroaches were acclimated to the environment of the experimental arenas by restricting them to a piece of plastic tubing which was placed in the untreated area for 5 min. Insects were released by carefully lifting the tubing in each arena. Bioassays were conducted under ambient temperature (25 ± 2°C) and relative humidity (40 ± 10%).

Example 6

[00124] Repellency assays with aged residue were performed. The two best-performed fresh resin fractions (“E” and “J”) were chosen to evaluate their residual repellency effect on cockroaches. Each fraction was tested as a fresh and an aged form at the same time. The assays also included fresh and aged residues of java oil, and acetone. Coconut fatty acid was added to this experiment as it has been reported to have longer residual effect than other proven repellents such as DEET. This longer-lasting effect may be beneficial for comparison to a positive control if java citronella oil loses potency over time. Tents (i.e. , harborages) in this experiment were treated as in the experiment to produce the aged residues, and the treated tent was left under the fume hood for 7 days. After the 7-day aging period the residual repellency assay was conducted with five residual and five fresh materials with two replicates in each run (20 arenas in total). 24 replicates were obtained from 12 runs for each material.

Example 7

[00125] Behavioral test results were performed, including distance to zone, duration in zone, number of visits, latency, and velocity. Repellent activity of the three guayule resin fractions from solvent extraction was assessed. Cockroaches significantly avoided the areas treated with java, resin (whole), terpene-rich, and lipid-rich fractions. As the resin is a complex mixture of compounds, including low-molecular weight rubber (“LMWR”), its activity could be partly associated with the stickiness of LMWR. The cockroaches spent 2.2% and 1.3% of the total time in the treated areas with resin and terpene-rich fractions, respectively, which showed a significant difference in comparison with acetone (P<0.001), while the whole resin and lipid fractions still have a reasonable level of activity (P<0.05). When compared with that of negative control arenas (acetone), the elapsed time until the cockroaches’ first visit (latency) was significantly longer when the area was treated with the terpene-rich fractions (mean time=141±89 s vs. 79.1 ±27.6 s). The visitation rate of cockroaches to the treated area is another parameter indicating the repellency effect of a test material. The results showed the area treated with terpene-rich fractions and resin (whole) have a lower visitation rate, while the number of visits to the whole resin- and lipid-treated area was not significantly different from the negative control (mean=17.1 ±5.8 vs. 18.6 ±2.2, respectively; P> 0.05). The cockroaches spent significantly less time in areas treated with terpene-rich fractions (mean= 19.5 ± 3.9 s), whole resin (mean= 28.8 ± 3.6 s), and lipid fractions (mean- 33.0+6.0 s) compared with the negative control (mean= 54.0 ± 6.3 s).

Example 8

[00126] The repellent activity of nine guayule resin fractions from vacuum distillation was evaluated. After the preliminary test, the results showed that the chemical compounds in the resin have a reasonable repellency activity. In order to find the most active group of compounds, the resin was fractionated into nine cuts. The vacuum distillation method was chosen to minimize the chance of degradation of thermolabile compounds and increase the environmental and economic feasibility for large-scale application compared with conventional solvent extraction techniques. All the fresh fractions showed a very promising performance to repel insects over a 25 minute period. The average distance of the insect to the (-)control zone (treated by acetone) was approximately 3.26+0.18 cm, while this parameter for the positive control zone (treated by java oil) was 4.2±0.10 cm. All the vacuum distilled fractions in comparison with (+)control showed no significant differences (fraction B-J vs. Java, P>0.05), while significant differences were found between the fractions “E”, “F”, and “J", vs. other fractions (P<0.05). All the other fractions (I, G, H, C, and B) repelled the insect by an average of 4.2 cm. The whole resin (unfractionated) showed a reasonable repellency effect, but as the whole resin is a sticky material its activity could not only be associated with its chemical properties. As was expected, no significant difference was observed between the (-)control and fraction “residue”. This result showed that although all the fractions showed a very reasonable level of repellency, fractions E, F, and J exhibited the best performance among other fractions. The most abundant class of compounds in fractions “E” and “F” was sesquiterpenes (C ₁₅ H ₂₄ .), which have been known as one of the active repellent materials, while fraction J was rich in oxygenated sesquiterpenes. Visualizations of insect tracks and “heat maps” representing the time spent within certain areas of the arena were prepared. It was clearly shown that the insects spent most of their time (indicated by the color gradient) as far away from the treated zone (E, J, +control) as physically possible. The track and heat map of (-)control showed the normal movement of cockroaches in an untreated arena. Since the harborage and being on the edges are two strong attractive forces, it was expected that insects spent most of their time under the tent in (-) control arena.

Example 9

[00127] Tracking activity was measured for the cockroaches. A near-infrared (NIR) camera outfitted with a lens and infrared (“IR”) filter was used to record cockroach activity in the arenas under dark conditions. The camera was positioned 60-100 cm directly above the center arena. Light for the recordings was provided by two IR illuminators. Software was used to capture video images and to track the insect during 20-25 minute bioassays. Each arena was divided into two zones known as “treated” and “untreated”. Taking the data from the tracks, multiple variables were calculated. From the assays with resin fractions-treated arenas the following behavioral parameters were calculated: time until the first visit of cockroaches to the treated zone (latency), the number of visits to the treated zone (frequency), percent of time spent in the treated zone, distance to treated zone, and average velocity in the arena.

Example 10

[00128] Results of lasting effect of guayule resin fraction was determined. To evaluate the residual repellent activity of the tested materials the same repellency bioassays (dual and multi-choice) were repeated after 2 and 3 months. The activity of the resin fractions was confirmed by testing them in fresh form within a 25 minute period. The (action time or longevity) lasting effect is one of the most important characteristics of repellents. Since most botanicaily derived active ingredients are volatile compounds, they do not last long (short time of action). In this part, fractions “E” and “J", which showed the best performance in their fresh form, were selected for the lasting effect experiment. Also, two controls (java oil and acetone) were tested in both fresh and aged forms. A significant strong repellency was found from Fraction “J” after 7 days of application (J _aged vs. Act _aged , P<0.001). In the distance to zone parameter, the repellency of J _aged was almost the same as its fresh form (J _fresh : 4.2±0.03 cm vs. Jaged' 4.06+0.3 cm, P>0.05). In contrast, the Fraction “E” (like Java), which is predominantly constituted of volatile compounds (see Figure WEV.), lost all its activity after 7 days. (E _aged : 3.2±0.13 cm vs. Act _aged : 3.1±0.15 cm). No significant difference was observed between the coconut fatty acid and negative control in either fresh or residual form (Co.F vs. Act.F and Co.R vs. Act.R, P>0.05). Except for fresh java oil no significant difference was observed for latency, the number of visits, and velocity parameters. These results showed that fraction “J” due to its chemical composition (which will be discussed in the next section) not only has a strong repellency but also has a very long action time (at least 7 days), which makes it a perfect candidate for commercial development.

Example 11

[00129] GC-MS and fatty acid methyl ester (“FAME”) analyses were performed on 147 volatile organics in guayule resin fractions and were spectrally deconvoluted, and 87 were putatively identified. Annotation was performed by MS-DIAL using the overall match score between experimental and reference library spectra database for each fraction. All fractions were qualitatively and semi-quantitively compared to the whole unfractionated resin. Fractions B & C were proportionally higher in 45 terpenes and terpenoids with a >99% summed relative abundance. Fractions E-l contained a higher sesquiterpene content where 42 individual compounds were identified with a summed relative peak abundance > 60% with fractions G-J having been in excess of 90% sesquiterpene content. Fractions B & C were enriched for alpha and beta pinene, ortho cymene, sylvesterene, and beta phellandrene. Fractions G-J were enriched for alpha isocomene and alpha eudesmol as showed by the results of GC-MS and FAME analysis of guayule resin fractions. Fractions “A" and “L” represent whole resin and distillation residue, respectively. Fractions B-J are 9 cuts obtained from distillation experiments. As was expected the compounds were distributed based on the boiling temperature in a vacuum distilled fraction. First fractions are rich in light terpenes (monoterpenes) and as the distillation moves forward with increasing temperature terpenes with higher carbon numbers come out. Monoterpenes are the dominant group of compounds in fraction α- and β-pinene (C ₁₀ H ₁₆ ), are significantly abundant in this class. The bornyl acetate (C ₁₂ H ₂₀ O ₂ ) is the dominant compound among oxygenated monoterpenes. The majority of the total terpene content of fraction “E”-“G” are sesquiterpenes, of which E- caryophyllene (C ₁₅ H ₂₄ ) is one of the most abundant compounds in this class. Fraction “J” which represented one of the best repellent efficacies, is rich in oxygenated sesquiterpenes (65.6% of total terpene content). The a-eudesmol (C ₁₅ H ₂₆ O) is the most abundant compound in this group, ranging from 2% in fraction “E” to 52% in fraction “J”. The total lipid and fatty acid content of the whole resin are 17.5 and 9.3 wt.%, respectively. Around 80% of the total lipid and 87% of the fatty acid composition of guayule resin constitutes C ₁₈ -C ₂₀ . Linoleic and linolenic are the most abundant fatty acids within the C ₁₈ -C ₂₀ range of fatty acids.

Example 12

[00130] The chemistry of active ingredients in guayule resin fractions was evaluated. From the chemical analysis of vacuum distilled fractions, the first fractions contain monoterpenes which is the most volatile group of terpenes in the resin composition. All the major compounds in this group such as α- and β-pinene, β-myrcene, α-phellandrene, and limonene have been widely reported as active ingredients of many essential oils used as insect repellents. Fraction “E” which was one of the repellents that had a low content of monoterpenes and monoterpenoids and high content of sesquiterpenes. The most abundant compounds in this group were E-caryophyllene and a-humulene, which have been known as strong natural repellents, a-eudesmol was the dominant compound in fraction “J”, and its activity is known against several insects. The high repellent efficacy of a-eudesmol against Turkestan cockroaches can be associated with the hydroxyl group. The OH-containing compounds positively affect the repellency activity against T. castaneum. The location of the OH group in the chemical structure can adversely affect the activity. For example, the OH linked to a tertiary carbon might suppress the activity against some species iike A. gambiae. From the structure! similarity analysis, it was shown that guayulin and argentatins, two major compounds in guayule resin, have physicochemical similarities with some known commercial repellents and/or insecticides. The characterization technique used in this study estimated that the boiling temperature of these compounds under the high vacuum condition proves that they most likely exist in the heaviest distilled cuts such as “I” and “J". The long-lasting effect of fraction “J” (at least 7 days) was attributed to high lipid and fatty acid content. The highly active terpenes (e.g., eudesmol) could have been encapsulated by lipid/fatty acid compounds. Encapsulating by lipids is one of the common methods to control the release of active compounds.

Example 13

[00131] Structural similarity analysis of guayule resin fractions was performed. To compare structural similarity between these four chemicals, the maximum common substructure (“MCS”) was calculated. A structure-based hierarchical clustering of molecules was used to quantitatively estimate chemical structural similarity. The chemical structure and physical properties of the 22 most abundant compounds in guayule resin were compared with 27 known active repellent/insecticide ingredients that are commercially available. A scatter plot showed the similarity of compounds using multidimensional scaling (MDS) with a similarity cutoff of 0.7. Mono-and sesquiterpenes were found in seven resin compounds. Citronella had a structural similarity with four other guayule resin compounds (beta-phellandrene, delta- cadinene, bornyl acetate, and myrcene). Interestingly MDS showed that guayulin, which is one of the unique compounds in guayule, had a similar structure to some of the known chemically synthesized insect repellents like DEET and pirimicarb. Argentatins (43,44), another most abundant class in resin, were structurally similar to propamocarb, IR3535, and picaridin. Fatty acid did not overlap with any known commercial insecticides/repellents. The compounds were also analyzed with the structure-based hierarchical clustering method and the molecular property descriptors were computed by the property prediction module. [00132] The preceding examples can be repeated with similar success by substituting the generically or specifically described components and/or operating conditions of embodiments of the present invention for those used in the preceding examples.

[00133] Note that in the specification and claims, “about” or “approximately” means within twenty percent (20%) of the amount or value given.

[00134] Although the invention has been described in detail with particular reference to the disclosed embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover all such modifications and equivalents. The entire disclosures of all references, applications, patents, and publications cited above and/or in the attachments, and of the corresponding application(s), are hereby incorporated by reference. Unless specifically stated as being “essential” above, none of the various components or the interrelationship thereof are essential to the operation of the invention. Rather, desirable results can be achieved by substituting various components and and/or reconfiguration of their relationships with one another.