MX2008005864A - Methods for determining the feeding habits of an animal - Google Patents

Abstract

A method of determining whether an animal has ingested a plant of interest is provided. The method comprises screening the animal for the presence of at least one indicator of a plant of interest.

Description

METHODS TO DETERMINE ANIMAL FOOD HABITS FIELD OF THE INVENTION The present invention relates in general to methods for determining the feeding habits and food history of an animal, and more particularly, to methods for determining the host plants of pests.

BACKGROUND OF THE INVENTION Transgenic crops resistant to pests develop continuously to allow increased crop yields while reducing the amount of pesticides required. However, the potential for pest resistance for transgenic crops is widely recognized, and the farming community is eager to establish protocols by which the emergence of completely non-susceptible pest populations can be significantly delayed or prevented. One way to reduce the rate at which pests develop resistance to transgenic crops is to ensure the presence of a refuge where susceptible pests are not exposed to the pesticide. In theory, the adult pests that emerge from the refuge environment will disperse and reproduce with any plague that emerges from the fields recombinant, and if any of the insects that emerge from the recombinant fields have developed a level of resistance to recombinant pesticidal proteins, the availability of that characteristic in subsequent generations will be diluted, reducing or delaying the start of the emergence of a race that It will be totally resistant to the recombinant plant. Shelter areas may consist of portions of the crop of interest that are untreated (ie, structured shelter) or another suitable crop and weed hosts of the pests (ie, alternate host or natural refuge). The evaluation of the refuge available for a pest that is capable of developing in multiple species of host silver requires some means to evaluate the portion of the insect population that exists in different potential hosts. In today's regulatory environment, obtaining approval from an appropriate regulatory agency for the commercialization of a recombinant plant requires that a percentage of the whole crop that is planted containing a recombinant characteristic be planted as a refuge from non-recombinant or non-transgenic crops in one farm base per farm. Shelter requirements increase the financial and labor costs of farmers, and are difficult to monitor. The added labor to plant and segregate the shelter and the equally inferior yields within the shelter as a result of insect infestation greater are a disincentive for the farmer to comply with regulatory requirements. In this way, there remains a need for methods to determine the feeding habits and food history of animals, and particularly pests, so that more effective areas of refuge can be determined or assigned. Also, it may be desirable to be able to analyze or take the fingerprints of an animal or population of animals in a way that easily identifies movement patterns and habits or food history.

BRIEF DESCRIPTION OF THE INVENTION A method is now provided to determine if an animal has ingested a plant of interest. The method comprises analyzing the animal for the presence of at least one indicator of a plant of interest. A high performance method is also provided to determine an animal's dietary history. The method comprises collecting a tissue sample from a plurality of animals, placing the tissue samples in individual wells of a multi-well plate, and analyzing each tissue sample for the presence of one or more indicators of a plant of interest. In addition, a method is provided to determine if an animal has ingested one or several plants of interest. The method includes collecting the minus one animal tissue; determine the fatty acid profile of the tissue; and comparing the fatty acid profile of the tissue with a fatty acid profile of an animal known to have consumed the plant of interest during its life cycle. There is still a method to determine if in the feeding stage an insect has ingested a plant of interest. The method comprises analyzing the insect for the presence of at least one indicator selected from the group consisting of gossypol, nicotine, nomicotin, cotinine, norcotinin, resveratrol, genestein, daidzein, glycitein, derivatives thereof, and combinations thereof. A method is also provided to determine if an insect has ingested a cotton plant in a feeding stage. The method comprises determining the relative amounts of C16: 1 and C18: 1 in the fatty acid profile of the adult insect. A method is also provided to determine whether a peanut plant has been ingested in an insect feeding cap. The method comprises determining the relative amounts of C16: 0, C18: 1, and C18: 2 in the fatty acid profile of the adult insect. A method is also provided to determine whether an insect has ingested a tobacco plant in a feeding stage. The method comprises determining the relative amounts of C16: 0 and C18: 3 in the fatty acid profile of the adult insect. A method is also provided to determine whether an insect has ingested a soybean plant in a feeding stage. The method it comprises determining the relative amounts of C16: 0, C18: 1, C18: 2, and C18: 3 in the fatty acid profile of the adult insect. A method is also provided to determine the natural refuge area for a pest in relation to a transgenic crop. The method comprises trapping pests from the vicinity of a transgenic crop, analyzing trapped pests for the presence of one or more indicators of at least one plant of interest, and determining the percentage of pest population that consumes a plant other than the transgenic crop. Additional features and benefits of the invention will be apparent to one skilled in the art from reading this specification.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a bar graph showing the fatty acid profiles of moths growing on cotton plants as determined in example 1. Figure 2 is a bar graph showing the fatty acid profile of moths growing in peanut plants as determined in example 1. Figure 3 is a bar graph showing the fatty acid profile of moths growing in tobacco plants as determined in example 1.

Figure 4 is a bar graph showing the fatty acid profile of moths growing in soybean plants as determined in example 1. Figure 5 is a graphic representation of the signal-to-noise ratio determined in the study of gossypol validation of example 1.

DETAILED DESCRIPTION OF THE INVENTION A method is now provided to determine if an animal has ingested a plant of interest. The method comprises analyzing the animal for the presence of at least one indicator of a plant of interest. Furthermore, before describing the invention, it is useful to understand the problem identified and addressed herein. The behavior patterns of the animal have a variety of commercial implications in agriculture, land use practices, conservation, real state, etc. The movement patterns are tracked extrinsically by radio transmitters, satellite technology, coding, etc., but there are difficulties. For example, radio telemetry is not practical for use with small animals or animals that migrate long distances. Satellite technology is prohibitive in terms of cost. Coding requires capture and recapture of some animals in a group, and those animals may not be representative of the group.

Stable isotope methods have also been used to track animal movement patterns. Stable isotopes are stable natural forms of elements with different nuclear masses. Stable isotopes are incorporated directly into animal tissues through the animal's diet. Although stable isotope methods do not depend on the recapture of previously captured animals, additional assumptions must be made. For example, differences in diet, location of forage, and metabolism, differences in climate and altitude, and differences in bedrock composition and heterogeneity in soil, invariably affect the isotope patterns in animal tissue. Gould et al. (2002) Proc. Nati Acad. Sci. 99 (26), 16581-16586 proposes a stable isotope evaluation as a way d? identify the host plants used by Helicoverpa zea larvae (bollworm) a crop pest. The composition of the stable carbon isotope (ratio of 13C to 12C, commonly reported as d13C) of C3 plants such as cotton and soybean is within the range of -20 to -32 0/00, and within a scale of -9 at -17 0/00 for C4 plants such as corn. Similarly, Bontemps et al. (2004) Proc. R. Soc. Lond. 271, 2179-2185 proposes the use of d13C to distinguish between host plants Ostrinia nubilalis (European drilling worm). However, the use of d13C is restricted for comparison between C3 plants and C plants, and may not be useful, for example, to distinguish moths reared as larvae in cotton and moths reared as larvae in soybeans.

In accordance with the present invention, applicants have discovered a method for determining whether an animal has ingested a plant of interest that can be applied to a variety of plants. The method generally comprises analyzing the animal for the presence of at least one indicator of a plant of interest. As used herein, an "indicator" of a plant of interest is any chemical compound that can be detected in or on an animal and which means that the animal or at a feeding stage of the animal ingested the plant of interest. . To be a successful indicator, the compound must be specific to the plant and not metabolized or predictably metabolized by the animal after ingestion. Preferably, the indicator is unique to the plant and causes a unique pattern change in the animal's biochemical composition. For example, in one modality the indicator is a biomarker. In another embodiment, the indicator is a chemical compound that is naturally found in the plant of interest, for example nicotine or gossypol. In another embodiment, the indicator is the result of human manipulation of a plant of interest, for example, a specific genetic marker for a transgenic plant. In even other modalities, the indicator is a chemical compound that is metabolized in a predictable manner by the animal after ingesting the plant of interest. As used herein, the term "intake" encompasses similar terms including, for example, consuming, eating, drinking, metabolizing, digesting and absorbing.

The analysis of an animal for the presence of an indicator may or may not require obtaining a sample from the animal. In modalities that require a sample, the sample may comprise tissue, hair, feather, saliva, sweat, tears, intestinal contents or excretions. In one embodiment, the method of the present invention comprises analyzing at least one tissue of the animal for the presence of an indicator of the plant. In a particular embodiment, at least one tissue of the animal includes the entire animal, for example, an insect. In other embodiments, the illustrative tissues may include skin, hair, feathers, wings, internal organs, blood, plasma, lymph, or the like. A tissue can be a complete organ, for example, a liver. Alternatively, the tissue sample can be obtained by biopsy. Usually, a tissue sample can be analyzed by a laboratory method or appropriate field test to determine the presence of the indicator of interest. Illustrative methods of analysis include protein extraction, fatty acid extraction, immunoprecipitation, DNA extraction, RNA extraction, PCR, Northern blot analysis, Southern blot analysis, Western blot analysis, elemental composition, chromatography, mass spectroscopy, immunostaining, microscopy confocal, and fluorescent microscopy. The methods of the present invention are generally useful for determining dietary habits or dietary history of a wide variety of animals including humans and non-humans, vertebrates or invertebrates. In several modalities, the animal is an insect, a fish, a bird, a reptile or a mammal. In addition, the animal can be domesticated or wild. In a particular embodiment, the methods of the present invention are used to determine the nutritional history of an insect, for example, a pest insect. The insects contemplated generally include any insect identified as a pest for an economically important crop plant. Examples of pest insects include, without limitation, northern corn rootworm, eastern corn rootworm, southern corn rootworm, cotton rootworm, tobacco rootworm, European wormworm, worm ejotero, cutworm, plant bug and bug. Likewise, the plant of interest can include any plant that is consumed by an animal directly, or that is consumed indirectly through the food chain. In one embodiment, the plant of interest is an agricultural crop plant, for example, cotton, corn, cannula, ear, tobacco, soybean, peanut, sunflower, rice, alfalfa or wheat. In another modality, the plant of interest is a fruit or tree plant. In another modality, the plant of interest is a plant plant. In yet another embodiment, the plant of interest is selected from the group consisting of sugarcane, cocoa plants and coffee plants. As described above, suitable indicators include any chemical compound that can be detected in or on an animal and which means that the animal or that at a feeding stage of the animal This animal ingested the plant of interest. In embodiments where the plant of interest is a crop plant, suitable indicators can be selected from the group consisting of a fatty acid, a tocopherol, a sugar, a flavonoid, nicotine, nornicotine, cotinine, norcotinin, gossypol, a protein vegetable, a mineral, a secondary metabolite of plant, a derivative thereof, and a combination thereof. Illustrative flavonoids include anthocyanins, flavanols, flavonones, flavonols, flavones, and isoflavones. Exemplary isoflavones include genestein, daidzein and glycytein. Illustrative tocopherols include RRR-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, tocotrienols, alpha-tocotrienol, and delta-trienol. Illustrative sugars include glucose, fructose or maltose. Exemplary fatty acids include C: 16: 0, C16: 1, C18: 0, C18: 1, C18: 2, and C18: 3. Illustrative minerals include calcium, iron, magnesium, phosphorus, potassium, sodium, zinc, copper and manganese. Exemplary plant proteins include resveratrol. Secondary metabolites of illustrative plants include gossypol, and an alkaloid. Illustrative alkaloids include solanine. In a particular embodiment, when analyzing an animal for the intake of cotton, gossypol may be an adequate indicator. Gossypol is a polyphenolic aldehyde pigment present in the seeds, root bark, and subepidermal glands of plants of the genus Gossypium, and in particular, cotton. Rojas et al., (1992) Environmental Entomoloqy 21 (3), 518-526 discusses the distribution of gossypol and metabolites in Heliothis virescens (tobacco worm), a culture pest. Heliothis larva feeds on crop plants including cotton. The authors report that the adult Heliothis moth contains 2.4% of the total gossypol ingested by the Heliothis larva. Gossypol is uniquely related to lysogenic cotton glands (Gossypium spp.) And related plants. The work by Rojas, et al., Showed that the adult moth of Heliothis virescens contained 2.4% of the gossypol ingested by the larval stage. The study indicated that all gossypol was a borderline form and no free gossypol was found. In this way the analytical methods that can determine the presence of a gossypol attached to the adult moth can allow the moths that develop in cotton to be discriminated against those that develop in other hosts. In another modality to determine if an animal has ingested a tobacco plant, the animal can be analyzed for the presence of nicotine or a nicotine derivative. Generally, nicotine is an adequate indicator for the intake of tobacco plants. However, since nicotine is relatively abundant in the environment from sources other than tobacco, nicotine derivatives that include nicotine metabolites may be preferred more as an indicator for tobacco use. Examples of nicotine metabolites include cotinine, nomicotin and norcotinin.

In even a further embodiment to determine if an animal has ingested a soybean plant, the animal can be analyzed for the presence of one or more isoflavones. Suitable isoflavones include, for example, genisten, daidzein or glycitein. In another embodiment to determine if an animal has ingested at least one plant of interest, the method comprises harvesting at least one tissue from the animal; determine the fatty acid profile of the tissue; and comparing the fatty acid profile of the tissue with a fatty acid profile of an animal known to have consumed the plant of interest during its life cycle. Generally, the fatty acid profile is determined by contacting the tissue sample with a solvent to extract fatty acids from the tissue sample. The extracted fatty acids are then transesterified to produce methyl esters of fatty acid which can further be separated and detected to determine a fatty acid profile for the tissue sample. In some modalities, the presence of a fatty acid will determine if an animal has ingested a plant of interest. In other embodiments, two or more fatty acids in combination will determine whether an animal has ingested a plant of interest. In even other modalities, the fatty acid profile will determine if an animal has ingested a plant of interest. The fatty acid profile can be any ratio of one or more fatty acids relative to the total fatty acids measured. For example, it has been found that the relative amounts of C16: and C18: 1 in the fatty acid profile of the animal can determine whether a animal consumed cotton plants. In addition, the relative amounts of C16.0, C18: 1, and C18: 2 in the acid profile of the animal are indicative of peanut plants; the relative amounts of C16: 0, C18: 1, C18: 2, and C18: 3 in the fatty acid profile of the animal are indicative of soybean plants; and the relative amounts of C16: 0 and C18: 3 in the fatty acid profile of the animal are indicative of tobacco plants. In some embodiments, the method comprises first analyzing an animal tissue for the presence of an indicator of a plant of interest, and second determining the fatty acid profile of the tissue and comparing the fatty acid profile of the tissue with an acid profile. indicator of the power in the plant. In other embodiments, the method comprises first determining the fatty acid profile of the tissue and comparing the fatty acid profile of the tissue with a fatty acid profile of an animal known to have consumed the plant, and secondly analyzing the tissue for presence of a plant indicator. In a particular embodiment, the methods of the present invention are configured to provide a high throughput method for determining the feeding characteristics of an animal. The method generally comprises collecting tissue samples from a plurality of animals and placing the samples in individual wells of a multiwell plate. Each sample in the multiple well plate is then analyzed for the presence of at least one indicator of a plant of interest or for determine the fatty acid profile of the tissue samples as described above. The methods and concepts described herein to track the feeding habits or nutritional history of an animal have multiple applications. In a particular embodiment, the methods of analysis described herein can be used to determine the natural areas of refuge of pests in relation to a transgenic crop. Said method comprises trapping pests from the vicinity of a transgenic crop; analyze trapped pests for the presence of one or more indicators of at least one plant of interest; and determine the percentage of pest population that consumes a plant other than the transgenic crop. Alternatively or additionally, the method may comprise collecting at least one tissue from trapped pests; determine the fatty acid profile of the tissue; and comparing the fatty acid profile of the tissue with a fatty acid profile of a pest known to have consumed the plant of interest during its life cycle before determining the percentage of the pest population that a plant other than the transgenic crop consumes. In addition, the product developers, scientists and regulatory authorities involved in determining pest refuge areas can use the information regarding food habits and dietary history of the pests to determine if the natural refuge area for these pests is sufficient to replace in part or completely the need of farmers to plant structured shelters for transgenic crops of interest in their farms, delaying or thus significantly avoiding the development of resistant pest populations while maximizing crop yield.

EXAMPLE The following example is merely illustrative in nature and should not be interpreted in a limiting sense. This example demonstrates a method of the invention for determining whether an animal has ingested a plant of interest. The experiment involves feeding moths of tobacco worms in the stage of larvae in tobacco, cotton, soybean or peanut plants. After the metamorphosis, the adult moths were analyzed for their fatty acid profile. The profiles were used to determine differences in lipid fatty acid profiles for each of the host plant species. The moths were then analyzed for the presence of cotinine (a nicotine metabolite) to determine if the moths developed in tobacco. Finally, the moths were analyzed for gossypol to determine if the moths developed in cotton. Although the analyzes in this experiment were completed in sequence, it is important to note that the analyzes can be carried out in any order independently of each other.

Fatty Acid Extraction Adult moths were harvested, freeze dried, weighed and individually placed in 2 mL wells of a 96-well plate. The moths were then crushed by adding a glass sphere to each well, covering the plate and placing the plate in a shredder as described, for example, in the U.S. patent. No. 6,880,771, which is incorporated herein by reference. During grinding, the shredder shook the plate at 800 rpm for 60 seconds. After triturating, diethyl ether (1 mL) was added to each well. The new plate was capped and swirled for 15 minutes to extract fatty acids from the moth matrix in the diethyl ether. The fatty acids in the ethyl ether were placed in a 2 mL well in a new 96-well plate for transesterification. The original 96-well plate containing the moths was placed under a dry stream of nitrogen to remove residual ether, and was subsequently capped and stored at -20 ° C for final determinations of continine and grossipol.

Transmethylation of extracted fatty acids Methyl acetate (20 μL) and sodium methoxide (40 μL) were added to each well containing the fatty acid in the diethyl ether. The new plate was swirled for 30 seconds and the solution allowed to stand at room temperature for 10-40 minutes. Next, diethyl ether saturated with oxalic acid (30 μL) was added and the plate was swirled for at least 20 seconds. The ether solution was then removed using a dry stream of nitrogen. After drying, hexane (1.5 mL) was added to each well and the plate was vortexed. One sample (1 mL) from each well was transferred to a bottle of the automatic analyzer for analysis by gas chromatography.

Gas chromatography and mass spectrometry conditions Gas chromatography comprised a DB-FFAP column, meters long, 0.25 mm in diameter, and in film thickness of 0.25 microns. The inlet temperature was 250 ° C and the injection was set for a fractional injection (6: 1 ratio). Each sample (1 μL) was injected into helium as the carrier gas at a flow rate of 0.8 mL / minute. The column was operated at a temperature of 85 ° C for 30 seconds, then approached by a ramp at 150 ° C at a speed of 25 ° C / minute, then approached by a ramp at 250 ° C at a speed of 17 ° C / minute. The column was then maintained at 250 ° C for 3 minutes. The detector was a detector sensitive to the impact mass of electrons, with mass detection fixed between 60 and 350 m / z. The areas integrated with fatty acid were obtained for C16: 0, C16: 1. C18: 0, C18: 1. C18: 2, and C18: 3.

Cotinine analysis The crushed moths that remained in the 96-well plate wells were analyzed for the presence of cotinine in accordance with the following: Cotinine Extraction An extraction solution was prepared by adding acetic acid (50 mL) to a 1000 mL volumetric flask followed by the addition of methanol (200 mL). Deionized water was added to the flask to bring the total volume to 1000 mL. 40% NaOH was added to the extraction solution to increase the pH above 11. The extraction solution (1 mL) was added to each well containing a crushed moth. Then, deuterized cotinine (20 μL) was added as an internal standard. The plate was then covered by placing the parafilm on the upper part of the plate subsequently pressing the cap on the plate on the parafilicle. The capped plate was first vortexed for 15 minutes, then centrifuged. The liquid layer of each well was removed and added to 8 mL bottles. A second volume of extraction solution (1 mL) was added to each well, the plate was swirled for 5 minutes, and then centrifuged. The liquid layer of each well was added to the previous one in the respective 8 mL bottle. To each 8 mL flask, 40% NaOH (150 μL) was added, followed by deionized water (4 mL). The 96-well plate containing the residue of moths is dried under a dry stream of nitrogen and stored at -20 ° C until further analysis. A solid phase extraction (DVB SPE) of divinyl benzene 100 mg was used to remove the cotinine from the extraction solution. DVB SPE was prepared by washing the column with ethanol (2 mL) followed by deionized water (2 mL). Subsequently, the cotinine in extraction solution was passed through the column followed by additional deionized water (1 mL). The column was dried for 5-30 minutes as air passed through the column. Subsequently, the column was washed with 20% methanol in ether (3 mL) to elute cotinine from the column. The methanol / ether was removed from the cotinine using a dry stream of nitrogen. The cotinine was resuspended in methanol / ether (150 μL), and the sides of the column were washed to avoid loss of any sample. The samples were placed in vials of the automated analyzer for GC / MS analysis.

Gas chromatography and mass spectrometry Gas chromatography comprised a DB-5 column, 15 meters long, 0.25 mm internal diameter and a film thickness of 025 μm. The inlet temperature was 285 ° C and the injection was set for fractionated injection / no flow division (ratio of 6: 1). One microliter of each sample was injected with helium as the carrier gas and a flow rate of 21.1 mL / minute. The column temperature started at 100 ° C, was maintained at that temperature for 0.1 minutes, approached by a ramp at 175 ° C to a speed of 40 ° C / minute, followed by a ramp approach of 30 ° C / minute at 300 ° C. The mass spectrometer used was a Leco flight time Pegasus III, with electron impact ionization energy of 70 eV and solvent delay of 50 seconds. The sweep scale was 50-210 m / z with 15 sweeps per second. The ion source temperature was 200 ° C. The quantification of cotinine in the samples was determined by measuring the ratio of the area from m / z 176 to the area of m / z 180, where m / z 180 was the standard deuterized cotinine. The cotinine standard in each sample was used to determine the retention time. The cotinine data were measured in parts per billion, with a detection limit of 1 part per billion.

Gossypol analysis The rest of the crushed moths in the 96-well plate were used for the presence of gossypol in accordance with the following: Extraction of gossypol Most of the gossypol found in moths was metabolized and bound to the protein. To extract gossypol in the bound form, the complex can be derivatized by creating a Shiff base with aniline that forms dianilino-gossypol.

The dianilino-gossypol can then be isolated from the moth matrix using DVB SPE. Both steps are described in detail below.

The derivatizing agent was prepared by mixing aniline (1 mL), glacial acetic acid (5 mL), and dimethylformamide (44 mL). The derivatizing agent can be stored at 4 ° C for one week. The deuterized derivatizing agent was prepared by mixing deuterated aniline (0.1 mL d5-aniline), acetic acid (0.5 mL) and dimethylformamide (4.4 mL). The deuterized derivatizing agent can be stored at 4 ° C for one week. The derivatizing agent (1 mL) was added to each well containing a crushed moth. The plate was covered with parafilm and a lid was pressed on the foil to seal each well. The plate was swirled for 1 minute, then the cap and seal were removed and the plate was covered with a foil. The plate was heated at 80-90 ° C for 1 hour in a hot well plate clamp oven or clamp. After removing from the heating system and cooling, the new plate was covered with the parafilm and capped to seal each well. The plate was centrifuged for 5 minutes at 2500-3000 rpm. DVB SPE was used to remove gossypol from the derivatizing agent. The 96-well DVB SPE plate was prepared by washing the columns with acetone (0.5 mL), followed by methanol (0.5 mL). The columns were then allowed to moisten with a solution comprising an equal mixture of water and DMF (0.5 ml). To avoid cross contamination, the cap and the parafilicle were carefully removed from the 96-well plate containing moths and the derivatizing agent. Next, water (0.5 ml) was added to each well.

Each sample was transferred to respective wells in the 96-well DVB SPE plate and allowed to pass through the column. Additional DMF (0.5 ml) was added to the original 96-well plate containing the moth residue, the plate was covered with parafilm and subsequently capped, vortexed for 1 minute, and centrifuged for 5 minutes at 3000 rpm. . After centrifugation, water (0.5 ml) was added to each well and the samples were transferred to respective wells in the 96-well DVB SPE plate. After the samples passed through the columns, the columns were rinsed with 20:80 water to methanol (0.5 mL) and followed by 5:95 acetone to methanol (0.5 mL). Subsequently, gossypol was eluted from the column with acetone (500 μL) in a 96-well plate of 2 mL or 1.5 mL. The acetone was removed from the samples and the samples were dried with a gentle stream of nitrogen. Samples were stored at -20 ° C until analysis by electro-spray ionization / mass spectrometry / mass spectrometry (ESI / MS / MS).

Determination of gsipol HPLC / ESI / MS / MS To each sample, acetone (200 μL) was added and the samples were mixed. Next, a solution comprising 5 μg / mL of deuterized derivatised dianilino-gossypol (20 μL) was added to each sample as an internal standard. The samples were capped and vortexed, subsequently transferred to a 96 mL 0.5 mL plate for analysis.

The HPLC used a short column (Zorbax Eclipse XBD-C18 commercially available from Agilent Technologies, Inc.) with a double solvent mobile phase. Solvent A had 10% water in methanol and solvent B was 2: 1 acetonitrile to acetone. The flow velocity was 0.25 mL / minute with a gradient as shown in Table 1.

TABLE 1 HPLC solvent gradient The mass spectrometer was a Micromass Quattro detector Ultima LC / MS / MS by Waters, and contained a triple quadrupole mass spectrometer with negative electrospray ionization. Since HPLC did not provide the correct ionization matrix for ionization by negative diasiline-gossypol electroaspersion, 0.6% NH OH in methanol was introduced after the HPLC column at a flow rate of 0.05 mL / minute. Post-column mixing of NH4OH in MeOH with the HPLC eluent was achieved using a mixing connection T. In MS / MS mode, the first quadrupole was used to select 667.3 as the mass ratio a charge that represented the negative ion of dianilino-gossypol. In the second quadrupole, the mass to load ratio 667.3 was fragmented into a collision cell. The third quadrupole was used to select the fragment ion, monoanilino-gossypol, which has a mass to charge ratio of 574.2 as a result of the loss of an aniline. Detection of the second ion of the fragment provides improved specificity and increased signal-to-noise ratios. The semi-quantification of gossypol was determined by the ratio of the peak area of 574.2 m / z to the peak area of the dianilino-gossypol deuterized internal standard. These relationships were then compared with standards, preforms and results of control moths bred in the laboratory to determine if gossypol was present.

Results As described above, the tobacco worm moths that grew as larvae in tobacco, cotton, soybean, and peanut plants were analyzed for their fatty acid profile, presence of cotinine, and the presence of gossypol. To construct the fatty acid profile of each plant host, a total ion chromatogram was integrated and the area of each fatty acid (C16: 0, C16: 1, C18: 0, C18: 2, and C18: 3) was obtained and added together for a total. The fraction of a fatty acid out of the total is shown below in Tables 2 to 5. The comparisons of the four plants host show that each has a unique fatty acid profile that can be used to distinguish them from others.

TABLE 2 Fatty acid ratios for moths growing on peanut plants TABLE 3 Fatty acid ratios for moths growing on cotton plants TABLE 4 Fatty acid ratios for moths growing in soybean plants TABLE 5 Fatty acid ratios for moths growing on tobacco plants Soft modeling independent of class analogies (SIMCA) was used to develop a supervised classification model based on the fatty acid profiles of the moths growing in each of the four crop plants. The development of the SIMCA model generated a separate main component analysis model (PCA) for each of the four crop plants, or a class model. A new Unknown and new sample was classified with each PCA model and its class membership was determined by the minimum distance of the unknown sample from the PCA class model. See figures 1 to 4. The graph S / S0 versus H0 shows the distance from sample to model in relation to the average model distance (S / S0) in the abscissa and the use for each sample in the axis of the ordinate . Class boundaries are shown as horizontal and vertical lines at the 5% importance level. Unknown samples near the origin within both lines can be classified as elements of the class model. Samples outside these lines can be classified as not belonging to the class model. Figures 1 to 4 show that the fatty acid profile data can be used to classify the moth samples in accordance with the host plants with which the larvae were fed prior to metamorphosis. Table 6 shows the distance between the models. The greater the distance of the interemodelo, the greater the difference that exists between the classes. Typically, a model difference greater than 3 indicates class molars that are significantly different. With values on the scale of 200-45,000, class models are significantly different and can be used to classify new samples according to their class.

TABLE 6 Model distance between cotton, tobacco, soybean and peanut plants The validation tests for gossypol were run as two separate sets on two different days. Most insects grew in cotton. In the first group established on day one, insects fed on hobby, soybeans and peas were included for comparison. It was thought that this group would respond negatively to gossypol. On the second day, insects fed artificial diets were included as negative controls. On both days, the preforms and standards were also run as additional test controls. The results in Figure 5 are expressed as the signal-to-noise ratio recorded by the electrospray / mass spectrometer which gives the most consistent results. Other response parameters of the mass spectrometer, such as a peak area, can also be used. The signal-to-noise ratio, minimum, average and maximum, is shown for each of the treatment groups: insects raised with artificial diet, hobby, soybeans, peas or cotton (with the insects fed with cotton of the two days presented separately). ). A cut-off value of 12 for the ratio signal to noise was chosen as the value to determine if the gossypol was present or not (if it is greater, then the sample is positive, if it is less than or equal to, then it is negative). Using these criteria, insects fed cotton were always unidentifiable as positive for gossypol in the test. For moths that grew in cotton the signal-to-noise ratio was typically greater than 100, almost always greater than 30, and never less than 14. The minimum values on the two days were 14 and 18 respectively, with a total of more than 50 cotton-bred insects tested on each day. The signal to noise criteria depend on the total method and thus can vary from laboratory to laboratory. It is therefore important that a validation study can be conducted to set the criteria in a way that minimizes false negatives. Using the signal-to-noise criteria, the results of the validation study for the gossypol HPLC-MS test are shown in table 7. All moths fed cotton were found to be positive for gossypol. Moths that grew on other plants that do not contain gossypol had a false positive rate. Table 7 also shows that for moths that grew on plants without gossypol there was a false positive rate. In other words, the moths can be identified as grown in cotton when they did not. For example, 4 of the 28 moths fed alplan were identified as being fed with cotton. With a signal-to-noise set for gossypol detection in 12: 1, the test allowed for the successful identification of all insects from cotton (positive gossypol) but it will have a false positive rate of around 15%. That is, some number of insects that were not fed on cotton in a rough way will be identified as cotton.

TABLE 7 Results of the validation study

Claims (38)

NOVELTY OF THE INVENTION CLAIMS

1. - A method to determine if in the feeding stage an animal has ingested a plant of interest, the method comprises analyzing the animal for the presence of the at least one indicator of a plant of interest.

2. The method according to claim 1, further characterized in that the indicator is selected from the group consisting of fatty acid, a tocopherol, a sugar, a flavonoid, an alkaloid, a vegetable protein, a genetic marker, a mineral, a secondary metabolite, a derivative thereof, and a combination thereof.

3. The method according to claim 1, further characterized in that the indicator is selected from the group consisting of nicotine, nornicotine, cotinine, norcotinin and gossypol.

4. The method according to claim 1, further characterized in that the animal is a pest.

5. The method according to claim 1, further characterized in that the animal is an insect.

6. The method according to claim 1, further characterized in that the plant of interest is a crop plant.

7. The method according to claim 1, further characterized in that the plant of interest is a crop plant selected from the group consisting of cotton, corn, canola, cob, tobacco, soy, peanut, sunflower, rice, alfalfa and wheat.

8. The method according to claim 1, further characterized in that the animal is an acorn worm moth or a tobacco worm moth.

9. The method according to claim 1, further characterized in that the indicator is a flavonoid selected from the group consisting of anthrocyanins, flavonols, flavonones, flavonols, flavones and isoflavones.

10. The method according to claim 1, further characterized in that the indicator is tocopherol selected from the group consisting of RRR-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, tocotrienols, alpha-tocotrienol, gamma-tocotrienol and delta-trienol.

11. The method according to claim 1, further characterized in that the method further comprises: collecting a tissue sample from the animal; extracting fatty acids from the tissue sample; analyze the extracted fatty acids to determine a fatty acid profile of the animal; and comparing the fatty acid profile of the animal with a fatty acid profile of an animal known to have consumed the plant of interest during its life cycle.

12. The method according to claim 11, further characterized in that the method further comprises: transesterifying the extracted fatty acids to produce a mixture of methyl esters of fatty acid and separate and detect the mixture of methyl esters of fatty acid to determine the fatty acid profile of the animal.

13. The method according to claim 11, further characterized in that the method is performed in a high performance format comprising: collecting a tissue sample from a plurality of animals; place the tissue samples in individual wells of a multi-well plate; and analyzing each tissue sample for the presence of one or more indicators of a plant of interest.

14. The method according to claim 13, further characterized in that the indicator is selected from the group consisting of a fatty acid, a tocopherol, a sugar, a flavonoid, an alkaloid, a plant protein, a genetic marker, a mineral , a derivative thereof, and a combination thereof.

15. The method according to claim 13, further characterized in that the indicator is selected from the group consisting of nicotine, nornicotine, cotinine, norcotinin and gossypol.

16. A method to determine if an animal has ingested at least one plant of interest, the method comprises: collecting at least one tissue from the animal; determine the fatty acid profile of the tissue; and comparing the fatty acid profile of the tissue with a fatty acid profile of an animal known to have consumed the plant of interest during its life cycle.

17. The method according to claim 16, further characterized in that the method further comprises: putting in contact the tissue with a solvent to extract fatty acids from the tissue; transesterify the fatty acids to prepare a mixture of methyl esters of fatty acid; and analyzing the mixture of methyl esters of fatty acid to determine the fatty acid profile of the tissue.

18. The method according to claim 16, further characterized in that the plant of interest is a crop plant selected from the group consisting of cotton, corn, cannula, cob, tobacco, soy, peanut, sunflower, rice, alfalfa and wheat.

19. The method according to claim 16, further characterized in that the method further comprises analyzing the tissue for the presence of at least one indicator of a plant of interest.

20. The method according to claim 19, further characterized in that the method comprises determining the fatty acid profile of the tissue before analyzing the tissue for the presence of at least one indicator of a plant of interest.

21. The method according to claim 19, further characterized in that the indicator is selected from the group consisting of a fatty acid, a tocopherol, a sugar, a flavonoid, an alkaloid, a plant protein, a genetic marker, a mineral , a secondary metabolite, a derivative thereof and a combination thereof.

22. The method according to claim 19, further characterized in that the indicator is selected from the group consisting of nicotine, nornicotine, cotinine, norcotinin and gossypol.

23. - The method according to claim 16, further characterized in that the method comprises determining the relative amounts of C16: 1 and C18: 1 in a fatty acid profile of an adult insect to determine whether in the feeding stage the insect has ingested a cotton plant.

24. The method according to claim 23, further characterized in that the method further comprises analyzing a test tissue of the adult insect for the presence of gossypol.

25. The method according to claim 16, further characterized in that the method comprises determining the relative amounts of C16: 0, C18: 1, and C18: 2 in the fatty acid profile of an adult insect to determine whether in the feeding stage the insect has ingested a peanut plant.

26. The method according to claim 25, further characterized in that the method further comprises analyzing a test tissue of the adult insect for the presence of a protein-specific allergen.

27. The method according to claim 26, further characterized in that the protein-specific allergen is resveratrol.

28. The method according to claim 16, further characterized in that the method comprises determining the relative amounts of C16: 0 and 018: 3 in the fatty acid profile of an adult insect to determine if in the feeding stage the insect has ingested a tobacco plant.

29. The method according to claim 28, further characterized in that the method further comprises analyzing a test tissue of the adult insect for the presence of nicotine and / or a nicotine derivative.

30. The method according to claim 29, further characterized in that the nicotine derivative is selected from the group consisting of cotinine, norcotinin, nornicotine and other nicotine metabolites.

31. The method according to claim 16, further characterized in that the method comprises determining the relative amounts of C16: 0, C18: 1, C18: 2, and C18: 3 in the fatty acid profile of an adult insect for determine if in the feeding stage the insect has ingested a soybean plant.

32. The method according to claim 31, further characterized in that the method further comprises analyzing a test tissue of the adult insect for the presence of at least one isoflavone.

33. The method according to claim 32, further characterized in that at least one isoflavone is selected from the group consisting of genestein, daidzein and glycitein. 34.- A method to determine the natural refuge area for a pest in relation to a transgenic crop, the method includes: trap pests of the proximity of a transgenic crop; analyze trapped pests for the presence of one or more indicators of at least one plant of interest; and determine the percentage of population of pests that consume a plant different from the transgenic crop. 35.- The method according to claim 34, further characterized in that the indicator is selected from the group consisting of a fatty acid, a tocopherol, a sugar, flavonoid, an alkaloid, a plant protein, a genetic marker, a mineral, a derivative thereof, and a combination thereof. 36. The method according to claim 35, further characterized in that the indicator is selected from the group consisting of nicotine, nornicotine, cotinine, norcotinin and gossypol. 37. The method according to claim 34, further characterized in that the method further comprises: collecting at least one tissue from the collected pests; determine the fatty acid profile of the tissue; and comparing the fatty acid profile of the tissue with a fatty acid profile of a pest known to have consumed the plant of interest during its life cycle. 38.- The method according to claim 34, further characterized in that the pest is an insect.